BACT 211: ACID FAST STAINING

Acid-fast Stains

• Differential staining useful in identification of acid-fast bacilli (AFB)

• Higher affinity for primary stain (can’t uptake counterstain)

• Resists decolorizer

Aside from identification of AFB, what is the other use for acid-fast stains?

Preliminary diagnosis of Tuberculosis

N-glycolylmuramic acid

Found in mycobacterial cell wall

N-glycolylmuramic acid.

Also known as “Mycolic acid.”

N-acetylmuramic acid

Found in gram (+) bacterial cell wall

Pink/Red

This

Other Acid-fast Microorganisms

1. Tsukamurella

2. Gordonia

3. Nocardia

4. Corynebacterium

5. Rhodococcus

Parasites that are Partially Acid-fast

1. Cryptosporidium

2. Isospora

3% sulfuric acid

Alternative decolorizer for partially acid-fast parasites

Smear Positive

Most common method to confirm presence of TB

DOH-based Smear

• 2 samples (early morning → random)

• w/n 1 day

• ½ is positive

Book-based Smear

• 3 samples (all early morning)

• 3 consecutive days

• 2/3 is positive

Culture Positive

Löwenstein-Jensen Medium

• M. tuberculosis grows w/n 8 weeks or 2 months

• Cauliflower appearance

• Malachite green for sample decontamination

Petragnani

• High conc. of Malachite green

• Used for heavily contaminated samples

Rapid Diagnostic

• Molecular approach

• PCR-based

• Useful for identifying if patient is resistant to anti-TB meds

Purified Protein Derivative (PPD) Test

• “Mantoux test”

• For identification of latent TB, 48 hrs (3rd type)

• Uses ammonium sulfate (for heat killing)

• Skin reaction = probably positive

Bacillus Calmette-Guerin

• Vaccine that creates false positive result in PPD test

• For M. bovis

Sputum

Most common sample for acid-fast staining

Gastric aspirate

Sample used for infants

Cerebrospinal Fluid

• For identification of Tubercular meningitis (2nd type)

• Web-like pellicle appearance after refrigeration

3P’s Sample

• Pleural (lungs)

• Pericardial (heart)

• Peritoneal (abdomen)

Antibiotics used for TB

1. Rifampin

2. Isoniazid

3. Pyrazinamide

4. Ethambutol

5. Streptomycin

5-10 mL

Min-max volume of sputum sample required

More than 25 epithelial cells per low power field

This sputum sample is rejected because it has higher contents of saliva

Low Power Objective (LPO)

An objective lens used for sample viability

Oil Immersion Objective (OIO)

An objective lens used for reading smear preparation

Smear Preparation

• Coiling method (small circular motion)

• Size: 2 × 3 cm

• Thumb-shaped

Ziehl-Neelsen Method

“Hot method”

1. Primary stain w/ Carbol Fuchsin

2. Heat until boiling

3. Cool down for 5 mins

4. Wash off excess w/ water

5. Decolorize w/ acid alcohol (HCL) for 30 secs

6. Counterstain w/ Methylene Blue or Malachite Green for 30 secs

7. Wash off excess w/ water

Kinyoun’s Method

“Cold method”

• Primary stai: more lipid soluble & concentrated Carbol Fuchsin

• Uses Tergitol as mordant instead of heat

  • high phenol conc.

• Same procedure w/ hot method but is preferred

Baumgarten’s Method

• Mixture of 10 parts (95% alcohol) and 1 part (nitric acid)

• Differentiation of M. tuberculosis (stained blue) vs. M. leprae (stained red)

Gabbet’s Method

Uses Gabbet’s Methylene Blue as both decolorizer and counterstain

Pappenheim’s Method

• Uses Pappenheim’s differentiating stain

• Differentiates M. tuberculosis (stained red)vs. M. lacticola or M. smegmatis (both stained blue)

Modified-Acid Fast Stain

• Stain used for partially acid-fast parasites

• uses 3% sulfuric acid as decolorizer

Fite-Faraco’s

• Identification of M. leprae

• Uses “Hematoxylin” as counterstain instead of Methylene Blue

Auramine-Rhodamine

“Truant’s Method”

• Uses fluorescent dye and fluorescent microscope

• AFB = yellow appearance under black background

Spengler’s Method

• Used by colorblind individuals

• AFB = black appearance

Smear Result: 0

No AFB seen

Smear Result: +/-

1-2 per 300 fields

Smear Result: 1+

1-9 per 100 fields

Smear Result: 2+

1-9 per 10 fields

Smear Result: 3+

1-9 per field

Smear Result: 4+

Greater than 9 per field