See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/334977871

Ziehl-Neelsen’s Staining

Chapter · January 2018

DOI: 10.5005/jp/books/14206_11

CITATIONS READS
0 1,168

1 author:

Upasana Bhumbla
Geetanjali Medical University
50 PUBLICATIONS   16 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Practical Microbiology View project

Workbook for Practical Microbiology View project

All content following this page was uploaded by Upasana Bhumbla on 03 October 2019.

The user has requested enhancement of the downloaded file.

Chapter 10 Ziehl Neelsen’s Staining

Aim: To stain the given fixed smear of sputum by Ziehl Neelsen’s technique.
Principle: Ordinary aniline dye solutions do not readily penetrate the substance of the tubercle bacilli and therefore it is
not suitable for staining. By the use of powerful staining solution that contains phenol and with the application of heat,
which act as a mordant ,it can be made to penetrate the bacillus. Once stained the tubercle bacillus will withstand the
action of powerful decolorizing agents ,thus retains the stain .
Requirements: Strong carbol fuchsin, 20% H2SO4, 95% alcohol or acid alcohol, methylene blue solution, distilled water,
spirit lamp, compound microscope and cedarwood oil/liquid paraffin.
Composition of strong carbol fuchsin:
Basic fuchsin - 10 g
Absolute alcohol - 100 mL
5% phenol in water - 1000 mL

Procedure
Primary staining and mordanting:
The fixed smear is flooded with strong Carbol fuchsin for 5 minute and heated intermittently. Until the steam rises,
taking care to see that the stain does not boil and that the smear does not get charred. The smear is then washed well
with distilled water. The basic fuchsin in strong Carbol fuchsin , basic stain while carbolic acid acts as a mordant. On
heating, the mycolic acid in the cell wall of the acid fast organisms is liquefied and the basic stain imbibed by the
organism is fixed by the mordant. On washing, the smear is cleared of excess of the stain or any deposits.
•• Decolorisation:
The slide is then covered with 20% H2SO4 for 2–3 minutes and washed with water. This step is repeated till the smear
becomes colorless. With this, only acid fast organisms retain the basic stain, while the cells and other organisms are
rendered colorless. After that decolorise with 95% alcohol or we can use acid alcohol.
By decolorising with alcohol; saprophytes can be differentiated from Mycobacterium tuberculosis, as saprophytes are
only acid fast whereas mycobacterium tubercle bacilli is acid as well as alcohol fast
•• Counter Staining:
The slide is covered with methylene blue or 1–2 minutes, washed with water and blotted to dry.
Acid fast organisms remain pink while the other organisms and cells, take up the counter stain and turn blue.
•• A drop of cedarwood oil or liquid paraffin is placed on the stained smear.
•• The microscope is adjusted for increased light by raising the condenser and the smear is examined under the oil
immersion objective using the plane mirror.
Ziehl Neelsen’s Staining 51
 52 Workbook for Practical Microbiology

RNTCP Grading
Ziehl-Neelsen Staining Grading
Finding No of fields Grading Result
No AFB in 100 oil immersion field 100 0 Neg
1-9 AFB per 100 oil immersion field 100 scanty Pos
10–99 AFB per 100 oil immersion fields 100 1+ Pos
1-10 AFB per oil immersion field 50 3+ Pos
>10 AFB per oil immersion field 20 3+ Pos
 Ziehl Neelsen’s Staining 53

PRACTICAL 1
Stain the given smear of sputum by Ziehl-Neelsen’s technique and record your findings with a suitable diagram.

Observation: (In terms of: Color, shape, arrangement and background)

Inference
54 Workbook for Practical Microbiology
 Ziehl Neelsen’s Staining 55
Q1. Mention some acid fast organisms/structure.

Q2. What are the various specimens obtained in the laboratory for the diagnosis of tuberculosis?

Q3. Name the various media used for the isolation of Mycobacterium tuberculosis.

Q4. Mention the techniques used to concentrate mycobacteria in clinical specimens. When are they indicated?
56 Workbook for Practical Microbiology
 Ziehl Neelsen’s Staining 57
Q5. Describe Petroff’s method.

Q6. What is Koch’s phenomenon?

Q7. What is Mantoux test?

Q8. What is B.C.G.? How is it useful?

58 Workbook for Practical Microbiology
 Ziehl Neelsen’s Staining 59
Q9. What are the differences between Mycobacterium tuberculosis and Mycobacterium leprae?
a. Morphology :

b. Staining technique :

c. Generation time :

d.
Culture :

e. Laboratory animals used :

Q.10. What are the differences between Human Tb and Bovine Tb

Q.11 What are the various concentrations of H2SO4 used for various Mycobacteria.
 60 Workbook for Practical Microbiology

View publication stats