Certifiable Skill Exercise
Ziehl-Neelsen staining
AIM:-To Stain the given fixed smear of sputum by Ziehl Neelsen's technique
Requirement:- Strong Carbol Fuchsin 20%H2s04,Methylene blue solution, Distilled water,
fixed smears, spirit lamp, Compound microscope and Cedar wood oil/liquid
paraffin.
PROCEDURE:-It consists of 3 Steps
Procedure ofAcid-Fast Stain.
1. Prepare bacterial smear on clean and grease free slide,
using sterile technique.
2.Allow smear to air dry and then heat fix. Cover the smear with strong carbol fuchsin stain.
3. Heat the stain until vapour just begins to rise (i.e. about 60 C). Do not overheat. Allow
the heated stain to remain on the slide for 5 minutes.
Heating the stain: Great care must be taken when heating the carbol fuchsin especially if
aining is carried out over a tray or other container in which highly fiammable chemicals have
collected from previous staining. Only a small flame should be applied under the slides using an
ignited swab previously dampened with a few drops of acid alcohol or 70% v/v ethanol or
methanol. Donot use a large ethanol soaked swab because this is a fire risk.
Wash off the stain with clean water.
Note: When the tap water is not clean, wash the smear with filtered water or clean boiled
rainwater.
5. Cover the smear with 20 for 5 minutes until the is
or smear sufficiently decolorized, i.e. pale
pink.
6. Caution: Acid alcohol is
fiammable, therefore use it with care well away from an open fiame.
7. Wash well with
clean water.
8. Cover the smear with malachite green stain for 1-2 minutes, using the longer time when
the smear is thin.
9.Wash off the stain with clean water.
Wipe the back ofthe slide clean; and place it in a draining rack for the smear to air-dry (do
not blot
.EXamine thesmearmicroscopically,usingthe 100 X oil immersion objective.
37
Exercise 4:
Competency
different causative agents of Infectious diseases ZN sta
by 7N
MI 1.2: Perform and identify the
Specific Learning Objectives:
Describe the principles of Ziehl-Neclsen stain.
smear provided, record the observations by draWing
Perform Ziehl-Neclsen stain on the
colors and interpret the findings.
ncat labeled diagram with appropriate
causing infectious diseases by Ziehl-
ldentify different acid fast organisms/structures
Neelsen Stain
Describe and apply RNTCP grading for suggesting therapy
Exercise 4.1:
1)Perform Zichl-Neelsen staining on the given heat-fixed sputum smear from a 42-year-old
of since 1
lady with cough with expectoration, evening rise of temperature and loss weight
month. Focus the smear under microscope, record your observations and interpret the results
Observations Aud fast baciu au sun {pink colourtd stghtycura
Inference Morphologcally relemb ung Mycobactouum tulerculosus
Examples M. loprat tuberculosts, HocaMd a , Aad fut
38
2) Describe the principle of Ziehl-Neelsen stain
Th opoky acud faltedneu i's based on presene o mycou
And
acid t the coll Nall
einding st eabo fuucun i h mycolie aud decolowuizahim
ith e0 Ha sO4 on't rmlease earbol fuckin from t coll
wall & auid fast orgonss mk rutaiM the rnd colourhinu thy au
Enlist various acid fast organisms/structures and the percentage (%) of decolourizer aud fas
3)
used for each type.
(sulfuric acid) decolourizer
Acid fast organisms/structuresPercentage (%) of
Acid fast organisms/structures Percentage (%) of decolourizer
Mycobactouum tubetculos
5
Mycobactouum oprat
Nocatdia
Aid fast forauete cyctopoa, 0.5
Lyptospoi dium cys torsospora,
Mioospondia)
4) What is Kinyoun's method? Write its applications.
Au
Ans .
Moditied method o ZN staining
is nown al wnyoun's cold Acid-
Di4fou from ZN stainin rom fut staini
s not equurnd Suwtabe fbr detechon o
Heah ycobacteua
Phenol Cancontrahon n Catbo fuchin - Pacolouuzaton wih 1
incrued tu o. 6 HCL nmethano used to
Puxakiom ocabol tuschin stainuy is
5) Write the principle of Ziehl-Neelsen stain
Mort
detection oocysts
cyctoora cxypto sporidrum
and 1sospora in stool spcinr
An The popety acid fad tness is based on the pvesene omcouc
acud in the s my cobac touum
coll wall
Pulmay stan, cabol fuuchin hinds to bycouc aud psank n
col Nall
Pecolouwi zakion M e0 sultuuC acid dou not release th pumany
stain from t cell ail an
y cobac teua tain tue ed colan el
fuuchin hance thy au called acid faut
Countxstaining ith methylen bue provides a contruting
bacugcound. for ob sunng th red stained mycobac toua
AU ycobactewa {MTe TM
39
au stong ty acd fast
. Leprae wealuly aud f t
Zichl-Neclscn stain
6) Record the observations of
M.tuberculosis
M.leprac
Nocardia Coccidian parasites
7) Explain RNTCP grading
Ans RAITCP- Ran'sed Nahonal Tubexculosts cantxol BogranmL
RATCA ronamid as Nahonol
Tubetuloss Eiminahon ProgrammL 202
Guudeinu for gradng zN stand
sputum Smeau
Humber oAFB SOn O1F toba screenid Crading Rukult
No AfB inioo oIF 00
-9 100 QIF Negahe
-99/100 OF 00 Scanty fosttive
-10/01F >10/ o1FF Postive
Pgsit
8) Describe the therapeutic
împlicatiön of RNTCP grading
Ans Moni totng the treatmant rupole o the
fahont
Asseliny t svouty the diseae
Ass aLting th nfc tiouunes the ahnt
Hghor u rad Mor is the nfuhiouneu
SMLA negahe ationt 10, D00 baca li/Ml sputu
au le nteethu 40
