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Acid-Fast Stain: A Technique for Bacterial Differential Staining, Study notes of Bacteriol, Exams of Bacteriology

Bethel University TennesseeBacteriology

Acid-Fast Stain: A Technique for Bacterial Differential Staining, Study notes of Bacteriology

Typology: Exams

2024/2025

Available from 11/23/2025

cecil27
cecil27 🇺🇸

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Acid-Fast Stain: A Technique for
Bacterial Differential Staining,
Study notes of Bacteriology
Acid-fast cells - correct answer*have a waxy cell wall
(and)
**mycolic acid* located in the cell wall makes these cells resistant to desiccation (drying)
& disinfectants
(also makes it difficult to stain these cells using traditional staining methods that are
water soluble)
Acid-fast genera include - correct answer*mycobacterium & some numbers of the
nocardia (a genus of weakly staining Gram-positive, catalase-positive, rod-shaped
bacteria)
Acid-fast staining - correct answer*a differential staining technique that differentiates
between acid-fast & non acid-fast bacteria
Compare & contrast the Ziehl-Neelsen & Kinyoun methods. - correct answer*Z-N: uses
steam to penetrate cell wall --> primary stain (carbolfuchsin) ---> decolorized w/ acid -
OH ---> counterstained w/ methylene blue for non-acid fast cells
*Kinyoun: identical to Z-N method - uses carbolfuchsin ---> acid -OH ---> methylene
blue, but does not require the slide to be steamed
Differential Staining - correct answer*using multiple stains to better differentiate
between different microorganisms or structures/cellular components of a single
organism
How long should the slides steam for in the Z-N technique? - correct answer*for at least
5 minutes to dye the acid-fast cells
(slide should not dry out; also cool slides before decolorizing)
Kinyoun method - correct answer*identical to Z-N method - uses carbolfuchsin, acid -
OH & methylene blue, but does not require the slide to be steamed
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Acid-Fast Stain: A Technique for

Bacterial Differential Staining,

Study notes of Bacteriology

Acid-fast cells - correct answerhave a waxy cell wall (and) mycolic acid located in the cell wall makes these cells resistant to desiccation (drying) & disinfectants (also makes it difficult to stain these cells using traditional staining methods that are water soluble) Acid-fast genera include - correct answermycobacterium & some numbers of the nocardia (a genus of weakly staining Gram-positive, catalase-positive, rod-shaped bacteria) Acid-fast staining - correct answera differential staining technique that differentiates between acid-fast & non acid-fast bacteria Compare & contrast the Ziehl-Neelsen & Kinyoun methods. - correct answerZ-N: uses steam to penetrate cell wall --> primary stain (carbolfuchsin) ---> decolorized w/ acid - OH ---> counterstained w/ methylene blue for non-acid fast cells Kinyoun: identical to Z-N method - uses carbolfuchsin ---> acid -OH ---> methylene blue, but does not require the slide to be steamed Differential Staining - correct answerusing multiple stains to better differentiate between different microorganisms or structures/cellular components of a single organism How long should the slides steam for in the Z-N technique? - correct answerfor at least 5 minutes to dye the acid-fast cells (slide should not dry out; also cool slides before decolorizing) Kinyoun method - correct answer*identical to Z-N method - uses carbolfuchsin, acid - OH & methylene blue, but does not require the slide to be steamed

the primary stain reagent contains higher concentrations of phenol (lipid solvent) & basic fuchsin than in the Z-N reagent Kinyoun method procedures - correct answerRepeat steps 1-3 above then, cover the slide with Kinyoun carbol fuchsin; stain for 3 minutes rinse with water; decolorize with acid -OH until -OH is colorless rinse with water; counterstain with methylene blue for 2 minutes rinse with water; blot dry List the two methods to stain acid-fast bacteria. - correct answer(1) The Ziehl- Neelsen method, which uses a steaming water bath to facilitate staining of the cell (2) The Kinyoun method, which uses more concentrated reagents & does not require steam (cold method) Mycobacteria - correct answersticky due to mycolic acid in their cell walls Organisms - correct answerMycobacterium smegmatis (acid-fast) Staphylococcus aureus (non acid-fast) Patient F culture (both) Pathogenic mycobacterial species includes - correct answerthe causative agents of tuberculosis (TB) & leprosy Phenol - correct answera lipid solvent increasing the concentration of phenol increases the solubility of the cell wall or dissolves the waxy cell wall & allows the basic fuchsin to penetrate the cells the higher concentrations permits the staining of the cells at benchtop temperatures Procedure for Z-N - correct answer(1) Label slide with the 2 organisms and the mix specimen; place 3 drops of water in each slide (2) Aseptically remove S. aureus & place in appropriate places; do the same with M. smegmatis; Patient F *(3) Air-dry slides; then, heat-fix

Why do we place a paper towel over the smear? - correct answerto decrease spattering & to prevent crystals from forming directly on the smear when steaming (this step is not necessary if using the Kinyoun staining technique) Why is acid-fast clinically important? - correct answerlook for tb or leprosy some mycobacterium are pathogenic identify acid-fast bacilli Why was it important to steam the slides in the Ziehl-Neelsen method? - correct answerto "melt" the waxy cell wall (mycolic acid) to permit penetration of primary stain carbol fuchsin Ziehl-Neelsen method - correct answer(1) entails steaming heat-fix slides over boiling water with the primary stain, carbolfuchsin (2) the heating "melts" or increases the fluidity of the waxy cell wall (mycolic acid) of acid-fast cells & permits the penetration of the primary stain (3) after staining, the slide is cooled - as the slide is cooling, the cell wall firms up & loses it fluidity... trapping the stain in the cell wall (4) Smears are decolorized with acid -OH, cells retaining the primary stain after decolorization are called acid-fast (meaning the bright-red fuchsia color remains fast in the cells) (5) The slide is then counterstained with a secondary stain, methylene blue to dye the non-acid fast cells, which will appear blue