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Solutions:- Part 6 – Field Stain A and B, Preparation of the solution

August 15, 2022CytologyLab Tests

Field Stain A and B

Field stain consists of:

  1. Field stain A
  2. Field stain B

Thin and thick blood films are made and fixed in methanol for one minute.

Field Stain A (Dark violet color)

Using Commercially Available Powder.

  1. Field stain A commercially available powder = 5 grams
  2. Distilled water is heated to 80 °C or kept at 60 °C for 30 minutes. = 600 mL.

Procedure to make Field stan A from commercial powder:

  1. Field stain A (ready-made powder).
    1. Mix the powder into the water until it is dissolved.
    2. Filter the solution.
    3. Label the date.

Field stain A  from the reagents:

  1. Methylene blue (analytical grade) = 1.6 grams.
  2. Azur = 1 gram.
  3. Disodium dihydrogen phosphate anhydrous = 10 grams.
  4. Potassium dihydrogen phosphate anhydrous = 12.5 grams.
  5. Distilled water = 1000 mL.

Procedure for Field stain A preparation:

  1. Take Methylene blue (analytical grade) = 1.6 grams.
  2. Take Azur = 1 gram.
  3. Take Disodium dihydrogen phosphate anhydrous = 10 grams.
  4. Take Potassium dihydrogen phosphate anhydrous = 12.5 grams.
  5. Distilled water = 1000 mL.
  6. Mix both salts (3 and 4) in the water.
  7. Add 500 ml from above into a bottle containing glass beads.
  8. Add the stain powders (Methylene blue and Azur) and mix well.
  9. Then add the remainder of the solution.
  10. Mix well and filter into another clean bottle.
  11. Label the bottle Field stain A and also write the date.

Field Stain B (Orange color)

Using Commercially Available Powder:

  1. Field stain B commercially available powder = 4.8 grams.
  2. Distilled water is heated to 80 °C or kept at 60 °C for 30 min. = 600 mL.
  3. Mix well the field stain B until it is dissolved.
  4. Filter and label the date.

Field stain B reagents:

  1. Eosin(yellow water soluble) powder = 2.0 grams.
  2. Disodium dihydrogen phosphate anhydrous = 10 grams.
  3. Potassium dihydrogen phosphate anhydrous = 12.5 grams.
  4. Distilled water = 1000 mL.

Procedure for  Field stain B preparation:

  1. Mix both salts (2 and 3) in the distle water (1000 mL) to make the solution.
  2. Add 500 ml from above into a bottle containing glass beads.
  3. Add the stain powders Eosin and mix well.
  4. Then add the remainder of the solution.
  5. Mix well and filter into another clean bottle.
  6. Label the bottle Field stain B, and also write the date.

Staining Procedure for Field Stain A and B:

  1. Fill up two Coplin jars or wide-mouth bottles:
    1. Field Stain A (Blue stain).
    2. Field Stain B (Red stain).
  2. Make a blood smear on a clean glass slide, and it is dried in the air.
  3. Fix in methanol for one minute or get Spray ‘Easyfix’.
  4. Dry in the air.
  5. Dip fixed smear to Field Stain B (Red Stain) for 5  to  6 seconds.
  6. Wash in running tap water.
  7. Dip smear into Field Stain A (Blue Stain) for 10 to 30 seconds (adjust it).
  8. Wash in running tap water.
  9. Dry at air and see under oil immersion objective.
  10. The staining time may be adjusted.
Field stain A and B procedure

Field stain A and B procedure

Interpretation of the cells in Field stain:

Type of cells Color appearance
Red blood cells
Pink
Neutrophils granules Lilac color
Eosinophils Orange granules
Malarial parasites Deep red chromatin and pale blue cytoplasm

 

Field stain smear

Field stain smear

How to prepare thick and thin smears:

  1. Thick and thin blood smears can be made into two separate slides.
  2. Put two separate drops of the blood. For a thin smear, put a small drop and a large drop for a thick smear.
  3. Spread the drops by the cover glass.
How to make blood smear thin, and thick

How to make blood smear thin and thick

Note: The timings can be adjusted by staining a few slides.

Questions and answers:

Q1: What is the purpose of the thin and thick smears.

Q2: Will you fix the thick slides.

Answers:

Q1: Thick smears are made for the identification of malarial parasites. Thin smears are for the appreciation of the peripheral blood cells.

Q2: Thick smear does not need fixation.


Possible References Used
Go Back to Cytology

Comments

Leonce Reply
June 14, 2020

Sjaelewa kbsa

Dr. Riaz Reply
June 15, 2020

Thanks

Musa zakiyyu ladan Reply
August 6, 2020

Thanks.

Dr. Riaz Reply
August 6, 2020

Thanks, welcome.

Miraz Mahmud Reply
August 18, 2020

Sir pleased say
Which agur use in stain

Dr. Riaz Reply
August 18, 2020

Azur B is used.

Miraz Mahmud Reply
August 18, 2020

Whice agur use in stain

Dr. Riaz Reply
August 18, 2020

Azure B is used.

Miraz Mahmud Reply
August 18, 2020

Thanks

Aboobacker Reply
October 5, 2020

Very informative, thank you sir.

Dr. Riaz Reply
October 5, 2020

Thanks.

Praveen Reply
December 29, 2020

Why we are using field stain b for first staining

Dr. Riaz Reply
December 29, 2020

That is correct.

Ramlah Reply
April 17, 2021

Afternoon sir how can I use for stains during Field stain b

Dr. Riaz Reply
April 18, 2021

I have described the procedure, and you can follow those instructions. Rest you have to do trial on how many seconds or minutes are needed to get a good result. The time will be different for different solutions.

Precious Reply
May 4, 2021

Hello Dr. Riaz,
Does not filtering the constituted solution affect the staining of the samples?

Dr. Riaz Reply
May 4, 2021

If solutions are well mixed, I don’t think there will be any issue with staining quality after filtration.

DR mkembela Reply
May 20, 2021

But there is no conc of those field A and B

Dr. Riaz Reply
May 20, 2021

Field stains final preparations are the working solutions.

samson onesmo Reply
May 20, 2021

what is concentration of field stain specifically in working solution ?

Dr. Riaz Reply
May 20, 2021

Field stains preparations are the working solution, no need for further dilution. You have to adjust the timings for good stains, which will depend upon the pH of the water.

Festus Maloney Reply
August 2, 2021

Hello Dr. Riaz,
How would one diagnose a patient’s sample if malaria parasites are found in it?

If one was to use pluses to diagnose, and following this order: Scanty, +, ++, +++; where +++ is most severe and scanty least severe)

Dr. Riaz Reply
August 2, 2021

I think when malarial is positive, that is enough for the diagnosis. But need to see a thick smear which sensitivity is 2 parasite/1,000,000 uninfected RBCs.
Smears should be done every 6 to 12 hours for 3 consecutive days.
You can see this reference https://labtestsonline.org/conditions/malaria

Macdonald Mpando Reply
October 20, 2021

well explained,well detailed

Dr. Riaz Reply
October 20, 2021

Thanks.

suresh kumar Reply
November 11, 2021

SIR WHAT CAN I DO TO GET A CLEAR DIFFERENCE BETWEEN ACROSOME AND NUCLES OF HUMAN SPERMETAZOA WHEN STAINED WITH DIFF QUIK STAIN AS IAM GETTING MORE DARK BLUE COLOUR

Dr. Riaz Reply
November 11, 2021

I think if you dip in the alcohol (or spirit), The color will get lighter. I hope this technique will help you.

Marie Reply
December 29, 2021

Field stain A lyses the red cells and in this case, we need to see the red cells complete that’s why we use B first.

Dr. Riaz Reply
December 29, 2021

True. Thanks for the comments.

Emmanuel E Nkwocha Reply
February 10, 2022

Thank you so much.
This is very informative.

Dr. Riaz Reply
February 10, 2022

Thanks.

Shankar Reply
March 2, 2022

Thanks sir, for sharing the info on preparation of the stain,,, I was searching for it for so long…. Lucky I found your information…. Thanks and God bless.

Dr. Riaz Reply
March 2, 2022

Thanks a lot. I am glad that my website labpedia.net helped you.

H shankar rao Reply
March 3, 2022

Sir, is this ( field stain) the same as diff quick stain…. . I need it to study sperm morphology… Could you guide me pls..

Thanks

h shankar rao Reply
March 3, 2022

Sir, is this ( field stain) the same as diff quick stain…. . I need it to study sperm morphology…

Thanks

Dr. Riaz Reply
March 3, 2022

Both stains are variants of the Rowmanswky stain. You can easily stain the sperms.

naufal alief Reply
May 8, 2022

1. Sir , can i change this “Disodium dihydrogen phosphate anhydrous
& Potassium dihydrogen phosphate anhydrous” ingredient onto other buffer , because is not ready yet in my country ,
2. and what is the function of two ingredient ?
3. an then, may i adding the acetic acid in field stain B for sharpening analysis purpose
thank ou Dr Riaz

Dr. Riaz Reply
May 9, 2022

You can take Field stains A and B from the HD supplies, Merck/BDH Chemicals, or other reliable companies.. Then bring 500 mL distle water, heat it to boiling, then add the stain and mix it thoroughly. When it is cool, filter and keep it in the storage bottles.

Irene Reply
May 19, 2022

Thank you sir, your information is so educative

Dr. Riaz Reply
May 19, 2022

Thanks.

samson onesmo Reply
August 15, 2022

please can your send this document to me , i can used forteaching purpose
Thanks in Advance

samson onesmo Reply
August 15, 2022

how can preserve field stain A and B after preparation , in which bottle ? for how long

Dr. Riaz Reply
August 15, 2022

You can keep the Field stain in the colored (brown) bottle. After some time, filter the stain. These stains are stable indefinitely.

Shridhan Patil
November 21, 2022

Sometimes platelets are stained very light to unstained , what could be reason

Dr. Riaz
November 21, 2022

Please adjust the staining time. If platelets are overstained, then you can decolorize them with methyl alcohol.

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