Field Stain principle,composition,procedure and interpretation (test to detect malaria)

FIELD STAIN MLTGEEKS

Field’s Stain is a histological staining techniques and romanowsky stain, used for rapid processing of blood specimens and is used to stain thick and thin film (thick film vs thin film) of blood to detect spirochaetes, protozoa and other purposes.It consists of two differential stain.Field stain A which is methylene blue and Azure dissolved in a phosphate buffer solution.It is the basic component of the stain and Field stain B made up of  Eosin Y in a buffer solution which is the acidic component of the stain.

What is the principle of Field’s Stain

  • Field’s stains contain methylene blue and eosin.
  • These basic and acidic dyes induce several colours when applied to cells.
  • The fixator, methanol, does not allow any additional changes to the slide.
  • The basic component of peripheral white blood cell( cytoplasm) is stained with acid dye and described as eosinophil or acidophil.
  • The acid component that is nucleic acid of the nucleus takes on the basic dye and is stain blue to violet  called basophil.
  • The neutral components of the cells are stained by both dyes(Field’s stain A and B solution)

Composition of Field’s Stain

Since the staining process involve two different dyes that is field stain A and B hence the following are the ingredients used in the preparation of each staining solution

Field’s Stain A

Methylene blue1.300 gram
Potassium phosphate6.250 gram
Disodium hydrogen phosphate5.000 gram
Fresh distilled water550.000 ml

Field’s Stain B

Eosin1.300 gram
Disodium hydrogen phosphate5.000 gram
Potassium dihydrogen phosphate6.250 gram
Distilled water500.000 ml

What is the appearance of Field’s stain

  • Field’s stain A has is a dark violet coloured solution
  • Field’s stain B has is an orange coloured solution
field's stain A and B
field’s stain A and B

Field’s stain procedure

How to stain thick blood film using Field’s Stain

Red blood cells are lysed during this procedure. The diagnosis is based on the appearance of the parasite as it is used as a diagnostic test for malaria or malaria blood test and other protozoa. Compared to thin blood films (used to identify different types of malaria parasites), parasites appear more concentrated in thick blood films( denser under the microscope).

  1. Place a drop of blood on a slide and spread it over an area of about 1 cm2.( The film should be distributed so thinly that it appears transparent.
  2. Allow the film to air dry (Do not fix the slide)
  3. Flood or dip the slide in Field’s Stain A for 2-3 seconds.
  4. Wash or rinse the slide with distilled water (Agitate gently)
  5. Flood or dip the slide in Field’s Stain B for 2-3 seconds and wash with distilled water.
  6. Now air dry  the smear or slide and observe under microscope.
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NB : Care should be taken when examining thick blood films parasite slides, as clotted cells and platelets may be confused with malaria parasites.

Preferably examine the edges of the dried blood film. The edges of the film are often better than the center, where the film can be too thick or cracked. Investigate 200 microscopic fields of a stained thick film( approx. 10 min), before the slide is declared negative.

How to stain thin blood film using Field’s Stain

This modifies the original Field stain so that fixed thin films (thin film thickness) can be quickly stained. It is a useful method for the rapid identification of presumptive species of malaria parasites. This method is appropriate for Babesia sp., Borrelia sp., malaria parasites. Leishmania sp, and sp.

Malaria parasites shows adequate staining at all developmental stages.The identification of species of malaria parasite can be based on the size and shape of the different phases of the parasite and the presence of stippling( e.g. bright red dots) and fimbriation( i.e.The ends are ragged).

Malaria parasites may, however, be missed in a thin blood film if parasitemia is low.It is therefore recommended to examine a thick blood film.The red cells are approximately 6- 20 layers thick with a thick blood film, resulting in a larger blood volume being examined.However, Giemsa staining is always the method of choice for definitive differentiation of the species.

  1. After air drying  the thin blood film,immerse or fix the smear in methanol for 1 minutes.
  2. Flood or dip the slide in Field’s Stain A for 2-3 seconds.
  3. Wash it with distilled water,
  4. Flood or dip the slide in Field’s Stain B for 2-3 seconds and wash with distilled water.
  5. Now air dry  the smear or slide and observe under microscope.
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Microscopic Examination of field stained slide

After staining the smear with Field’s Stain,the slide is observed under microscope for staining characteristic using 100X objective with cedar wood oil or immersion oil.

NB :  that When examining thin blood smears for malaria, you should look for infected red blood cells and parasites inside the cells!

Field’s stain  staining characteristics under the microscope

Neutrophil granuleslilac
Eosinophilic granulesorange
Red cells pink
Nuclei blue
Malaria parasitesdeep red chromatin and pale blue cytoplasm.
Leucocytepurple nuclei and pale blue background
Red cells are lysedonly background stroma remains.
Field's stain microscopic observation
Field’s stain microscopic observation
Malaria parasite in field's stain
Malaria parasite in field’s stain

NB : Lysis of red blood cells may occasionally fail. White blood cells, blood platelets and malaria pigments can also be seen on a thick film. If present, schizocytes and gametocytes are also easily recognizable.

References

Protocols are adapted from http://www.southampton.ac.uk/~ceb/Diagnosis/Vol1.htm M. Arcari, A. Baxendine, C. E. Bennett (2000): A-Z Guide to Parasitology (Vol. 9 & 11)

  • World Health Organisation. Basic Laboratory Methods in Medical Parasitology (1991). ISBN 92 4 154410 4.
  • Greenwood, B.M (1997): What’s new in Malaria control? London School of Hygiene and Tropical Medicine, Keppel Street, London
  • Murray, PR, Drew, WL, Koyayashi, GS & Thomson, JH: Medical Microbiology. Mosby Books Inc., New York (1990)
  • Peters, W & Gilles, HM: Tropical Medicine & Parasitology. Wolfe Medical Publications Ltd.
  • Jeffrey & Leach: Atlas of Medical Helminthology and Protozoology. E & S Livingstone Ltd.
  • Ash, LR & Orihel, TC: Atlas of Human Parasitology. ASCP Press, Chicago.
  • Garcia, LS & Bruckner, DA: Diagnostic Medical Parasitology. Elsevier Science Publishing Co. Inc.
  • Muller, R & Baker, JR: Medical Parasitology. Gower Medical Publishing.
  • Smyth, J.D: Introduction to Animal Parasitology. Cambridge University Press (1994)
  • Snell, JJS, Farrell, ID & Roberts, C: Quality Control, Principles and Practice in the Microbiology Laboratory. Public Health Laboratory Service. ISBN 0 901 144 312.
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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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