(LML) Staining smears for leprosy

Leprosy Mailing List – July 29,  2014 

Ref.: (LML) Staining smears for leprosy

From:  Andrea Clapasson, Genoa, Italy

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Dear Pieter,

Thank you very much to you and your colleagues for the good job done with the leprosy mailing list.  I refer to your last mails about the important topic of staining of M. leprae.  Herewith are my comments.

There are different methods for staining but each method must be standardized by the single laboratory.

Several authors tell that they are using a Ziehl-Neelsen stain but this affirmation is too simple and, it is not clear.  Can a simple name (Ziehl-Neelsen’s method) create confusion?  Yes, it can, only after a carefully reading of the relevant papers it is possible understand the used procedure.  The methods reported as Ziehl-Neelsen are:

a.         Classical Ziehl-Neelsen’s method;  here the slide is warmed before to wash it by acid-alcohol solution;

b.         The Kinyoun staining, where the slide with sample is not flamed and it is known as cold Ziehl-Neelsen technique.  Personally I prefer this procedure (Fandinho FC and other Int J Lepr Other Mycobact Dis. 1990 Jun;58(2):389-91)

c.         Fite-Wade-Faraco

 

The critic steps are:

• concentration of the Primary stain
• volume of solution acid-alcool used in the destaining
• concentration of  acid in the destaining solution
• time exposition at Primary stain
• the critic step you can evocate for counter stain (concentration of blue methylene, its concentration, time ……)

 

If you read papers on this subject since 1915 you find so many changes and modifications one after the other that at times you lose the original work, new modifications not always provide relevant improvements and sometimes quality is lost.  Below are reported two papers that I regard as most valuable if you want to use Carbolfucsine for looking for M. leprae the reference article is the following one:

-           A comparison of the Ziehl-Neelsen and Kinyoun methods in staining smears from leprosy patients. Fandinho FC, Orsi-Souza AT, Salem JI. Int J Lepr Other Mycobact Dis. 1990 Jun;58(2):389-91.

Furthermore the following article it is of great interest:

-           Inefficiency of 0.3% carbolfuchsin in ziehl-neelsen staining for detecting acid-fast bacilli. Selvakumar N, Rahman F, Rajasekaran S, Narayanan PR, Frieden TR. J ClinMicrobiol. 2002 Aug;40(8):3041-3.

 

Herewith follows a description of the method I use in Genoa.

Cold Ziehl–Neelsen technique for slit-skin smear (SSS) and nasal swab (NS)

[Kinyoun staining]

1.         Cover the sample (skin smear o nasal swab) with primary stain (*), for 20 minutes;

2.         rinse gently with indirect stream of tap water, until the water flows off clear;

3.         decolorize each slide separately with 2,5 ml of solution of hydrochloric acid and ethanol or sulphuric acid and alcohol (**). This step is more critical of all procedure, because M. leprae is more easily decolourized than other mycobacteria, for example of M. tuberculosis. If duration of destaining is too long there are false negatives while if it is too short there are false positives.

4.         rinse with indirect stream of tap water;

5.         counter stain with methylene blue 1% (***), for 30 seconds;

6.         rinse the stain with indirect stream of tap water until the water flows off clear;

7.         allow slides to dry, away from sunlight;

8.         observe the slides under oil immersion.

 AFB appear red, while non-AFB other organisms and cellular materials appear blue.

 

(*) Primary stain:

a.         in a beaker previously weighed dissolve 6.75 g of basic fuchsin in 67.5 g absolute alcohol.

b.         Add 37.5 g of 5% aqueous phenol. [phenol solution: weight 5 g of phenol crystal and dissolve them in 100 ml distilled water (heating gently)]

c.         After add deionised water up to 675 g.

d.         Mix well and filter before use.

Prepare the solution with all components under the fume hood, using appropriate safety equipment (gloves, mask for dust and fumes). The prepared solution is transferred in dark glass bottle with screw cap (capacity one liter). Label bottle with name of reagent as well as preparation and expiry dates. Store at room temperature for six to twelve months.

 

(**) Decolorizing reagent: 95ml ethanol 96° and 1ml hydrochloric acid 37% (fuming).  Important: you must always add acid, drop by drop, to solvent, not vice versa.

In countries where the acquisition of alcohol may be problematic, an aqueous solution of 23,75% sulphuric acid and 3% alcohol may be used as decolourising agent. This is prepared as follows: add 25 ml of 95% sulphuric acid slowly (not vice versa) to solution of 71,5 ml of distilled water and 3,3 ml of 90% denaturized alcohol.

 

(***) Counter stain: dissolve 1g methylene blue in 100 ml distilled water.

Many mycobacteria can survive and grow in nutritionally poor environments such as water puddles and even chlorinated tap water.  Environmental mycobacteria might be present in the tap water; boiled water does not solve the problem, you will kill them but they will appear again as AFB after staining. The water is a reagent and its quality is the most important thing, use purified or distilled water for your solution, not tap water, rain water or boiled water.

If it is possible include one positive and one negative control among the slides when you are staining, for the quality control of Ziehl-Neelsen reagents.

 

Andrea Clapasson

 

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LML - S Deepak, B Naafs, S Noto and P Schreuder
LML blog link: http://leprosymailinglist.blogspot.it/
Contact: Dr Pieter Schreuder <<editorlml@gmail.com

 

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