Department Of Microbiology

Tirunelveli Medical College Tirunelveli.

Acid fast staining

1. What do you mean by acid fastness?

Acid Fast Bacteria resist decolorisation with strong mineral acids.This is due
to the presence of mycolic acid in the cell wall. This property is called acid
fastness.
2. Name various acid fast organisms.
1. Tubercle bacilli,
2. Leprabacilli,
3. Atypical mycobacterium,
4. Nocardia spp.
5. Bacterial spores,
6. Oocysts of Cryptosporidium parvum and Isospora belli.
3. What are the factors which affect acid fastness of an organism?
 Age of colonies
 Medium on which growth occurs.
 Ultraviolet light
 Lipid content and integrity of the cell wall of the organisms.
4. What are the methods of acid fast staining?
 Hot method-Ziehl-Neelsen staining
 Cold method-
 Kinyoun acid fast stain
 Gabbotts method
5. Procedure of Ziehl-Neelsen staining?
 Step 1:Primary stain
 Smear is poured with strong carbolfuchsin(1%) and wait for 5 minutes.
 Intermittent heating is done by flaming the underneath of the slide until
the vapoursrise.
 Heating helps in better penetration of the stain.
  Rinse the slide with water until all free carbolfuchsin stain is washed
away.
 Step 2:Decolorization
 Flood the smear with 25% sulfuric acid over the slide
 Allow it to stand for 2-4 minutes.
 The slide is gently rinsed with tap water.
 Then rinse gently with tap water.
 Step 3:Counterstaining
 0.1% methylene blue is poured onto the slide and left for 30 seconds.
 Then the slide is rinsed gently with tap water and allowed to dry.
 Focus under oil immersion.
6. Interpretation of acid fast staining?
 Mycobacterium tuberculosis appears as long slender, straight or slightly
curved and beaded, pink colored acid fast bacillus.
 Other Non-Acid Fast organisms present in the smear and the background
take up the counter stain and appear blue.
7. Is there any difference in Ziehl-Neelsen staining procedure for Mycobacterium
leprae?
Mycobacterium leprae are weakly acid fast organisms.So Decolorization is
done with 5% sulfuric acid instead of 20% sulfuric acid.
8. Why heating of carbolfuchsin is necessary in Ziehl-Neelsen staining?
 Heating dissolves the waxy coat of mycobacteria cell wall and helps in
penetration of dye (strong carbolfuchsin) through surrounding the cell wall.
9. Why kinyoun acid fast stain method is known as cold method?
 The Kinyoun acid fast staining method is known as cold stain because the
staining is done without the use of heat.
 The high concentration of phenol with strong carbolfuschin as primary stain
serves as to dissolve the lipid material in the cell wall without heating.
10.Which method of staining is most useful for screening procedure?
Fluorescent dye staining.

11.What is the number of bacilli which must be present in the sputum for detection
by direct microscopy?
50000 to 100000 bacilli/ml.
12.What is the role of phenol in carbolfuchsin?
It acts as mordant.It also makes the cell surface easily penetrable for strong
carbol fuchsin by dissolving fats.

13.What is the concentration of sulphuric acid used for decolorisation of various

acid fast organisms?
Microorganisms Percent concentration of
sulphuric acid
1.Mycobacterium tuberculosis 20%
2.Atypical mycobacteria 20%
3. Mycobacterium leprae 5%
4.Cryptosporidium 1-5%
5.Nocardia spp. 0.5%
6.Bacterial spores 0.25-0.5%

14.Name another method used for detecting acid fast bacteria in a smear?
Fluorescent staining methods using fluorochrome dyes/stains such as Auramine
O and rhodamine is another method that can be used for detecting acid fast
bacteria in a smear.
The smear is focused under low power in fluorescent microscope .
By this method acid fast bacteria fluoresces bright yellow or orange against a
greenish background.
15.Name the various culture medias used to grow M.tuberculosis
a. Solid media:
i. Egg based-Lowenstein-Jensen medium
1. Dorset egg medium.
ii. Agar based-Middle brook7-H-10 medium.
1. Middle brook7-H-11 medium
b. Liquid media:
i. Dubos medium.
ii. Middle brook 7-H-9 medium
16.Name the most commonly used medium for growing M.tuberculosis.
Lowenstein-Jensen medium (LJ medium)-Selective medium for M.tuberculosis.
17.What are the constituents of LJ medium?

Composition Use
Beaten eggs Acts as a solidifying agent
Aspargine Source of nitrogen and vitamins.

Mineral salts Enhance growth and act as buffer

Malachite green Inhibits the growth of organisms other than

mycobacteria and also provides a color to the
medium.
Glycerol Improves the growth of human type of
M.tuberculosis.( it has no effect or even inhibit
M.bovis.)

18.Name the method used to sterilise LJ medium.

Inspissation.
19.How much time is required to grow M.tuberculosis on LJ medium?
4-8 weeks .
20.Describe the characteristics of M.tuberculosis colonies on LJ medium.
Colonies of M.tuberculosis are dry,rough,tough,and buff colonies .
21.Name few biochemical tests used to identify M.tuberculosis.
i. Niacin test
ii. Nitrate reduction test
iii. Neutral red test
iv. Susceptibility to thiophen-2 carboxylic acid.
22.Name the specimens collected for laboratory diagnosis of tuberculosis.
1.Pulmonary tuberculosis:
i. Sputum -Most common specimen.
 ii. Laryngeal swab
iii. Bronchial washings-Bronchoalveolar lavage
iv. Gastric washings in children-Resting gastric juice(RGJ)

2.Meningitis-CSF
3.Renal tuberculosis-Urine(three consecutive days morning samples)
4.Bone and joints tuberculosis-Aspirated synovial fluid.
5.Tissue-Biopsy of tissue.

23.What are the other methods for diagnosis of tuberculosis.

a. Direct microscopy-ZN staining of direct smear and concentrated smear.
b. Culture-LJ media.
c. Biochemical reactions
d. Animal inoculation
e. Sensitivity testing.
24.What are the advantages of concentration of specimens?
Concentration of a specimen is done for
i. Homogenisation of the specimen
ii. Decontamination i.e., to kill other bacteria present in the specimen.
iii. Concentration i.e., to concentrate the bacilli in a small volume without
activation.
25.Name the various concentration methods used in laboratory diagnosis of
tuberculosis.
1.Modified Petroff’s method-widely used technique.
Sputum is thoroughly mixed with equal volume of 4% sodium hydroxide ,
centrifuged and sediment is neutralized with phosphate buffer saline of pH 6.8.
2.N-acetyl-L-cysteine with 2% sodium hydroxide.
3.Pancreatin
4.Dilute acids

26.Name few rapid methods for diagnosis of tuberculosis.

1. Mycobacterial Growth Indicator Tube(MGIT)
2. Bact T/Alert 3D SYSTEM
3. PCR
4. CBNAAT-Cartridge Based Nucleic Acid Amplification Test.
27.What is tuberculin test (Mantoux test)?
It is delayed or type IV hypersensitivity reaction.
28.Name the reagent used for doing tuberculin test.
Purified Protein Derivative.

29.How is PPD injected and what is its dose?

PPD is injected intradermally and the dose is 0.1 ml
30.How much time does it take to read the results of tuberculin test?
It takes 48-72 hours after intradermal inoculation.
31.What is the positive tuberculin test?
Induration of 10 mm or more in diameter surrounded by erythema at the site of
inoculation.
32.Give the significance of positive tuberculin test.
a. Positive test indicates past infection with tubercle bacilli .
b. It does not indicate the presence of active stage of the disease.
c. The test is helpful in children under five years for indication of active
infection
d. The test becomes positive 4-6 weeks after BCG vaccination.
33.What is Koch’s phenomenon?
Robert Koch observed that guinea pig already infected with tubercle bacillus
developed a hypersensitivity reaction when injected with tubercle bacilli or its
protein.This observation is called as koch’s phenomenon.

34.What isKoch’s postulates?

Robert Koch had postulated that a microorganism can be accepted as the
causative agent of the infectious disease only if the following four criteria are
fulfilled as follows.
1. The microorganisms should be constantly associated with the lesions of
the disease.
2. It should be possible to isolate the organisms in pure culture from the
lesions of the disease.
3. The same disease should occur when the isolated microorganism is
inoculated into a suitable laboratory animal.
4. It should be possible to re-isolate the organism in pure culture from the
lesions produced in the experimental animals.
 5. Antibody to the causative organism should be demonstrable in the
patients serum.(Criteria added recently)

35.What are all the exceptions to koch’s postulates?

1. Mycobacterium leprae and Treponema pallidum.
They cannot grow in vitro.They can be maintained in experimental animals.
2. Neisseria gonorrhoeae:There is no animal model,but can be grown in vitro.
36.What is multidrug resistant (MDR) tuberculosis?
a. Multi Drug Resistant Tuberculosis refers to resistance to Rifampicin
and Isoniazid,with or without resistance to one or more antitubercular
drugs.
37.What is extensively drug resistant (XDR) tuberculosis?
 Extensively Drug Resistant Tuberculosis is due to Resistant to Isoniazid
And Rifampicin.
 M.tuberculosis strains which are resistant to any fluoroquinolone
 Resistant to at least one of the three injectable second line
drugs(Capreamycin,Kanamycin And Amikacin)
38.What is extremely drug resistant tuberculosis?
Extremely drug resistant tuberculosis is M.tuberculosis strains which are
resistant to all first line drugs and second line drugs.
39.Name the vaccine used for prophylaxis of tuberculosis.
BCG(Bacilli Calmette Guerin)
40.What type of vaccine is BCG?
Live attenuated vaccine.
41.What is the dose and route of inoculation of BCG vaccine?
0.1ml is injected intradermally.BCG vaccine should be given soon after birth.
42.What is the diluent used for BCG vaccine ?
Normal saline
43.Classify Atypical Mycobacteria.
a. Group I-Photochromogens-Produce pigments on exposure to light.
b. Group II-Scotochromogens-Produce pigment in dark.
 c. Group III-Non photochromogen-Do not form pigments
d. Group IV-Rapid growers- Produce colonies in 4 or 5 days.
44.Who classified atypical mycobacteria into four groups?
Runyon

45.Give examples of each group of atypical mycobacteria.

i. Group I-M.kansasii,M.simiae
ii. Group II-M.scrofulaceum,M.gordonae
iii. Group III-M.avium,M.intracellulare
iv. Group IV-M.chelonei,M.fortuitum.

46.Sputum collection:
a. Ask the patient to rinse the mouth with water.
b. Instruct patient to take a deep breath.
c. According to NTEP guidelines two specimens, one early morning and
other spot specimen are collected.
d. In case of delay, refrigerate the specimen.

47.RNTCP guidelines for grading of sputum smear.

No of AFB seen Oil immersion field Grading Result

to
be screened
No AFB in 100 OIF 100 0 Negative
1-9/100 OIF 100 Scanty Positive
10-99/100 OIF 100 1+ Positive
1-10/OIF 50 2+ Positive
>10/OIF 20 3+ Positive

48.What is RNTCP ?
Revised National TB Control programme
49.What is NTEP ?
National TB Elimination Programme