Experiment 6: The Acid Fast Stain (Kinyoun Method)

What type of staining technique is the acid fast method?

differential stain

used to detect presence/absence of mycolic acid in bact cell walls

why is this method useful in clinical microbiology?

used to identify species in the Mycobacterium genus bc some can be pathogens

1. M. tuberculosis - tubercolosis (lung disease)

2. M. leprae - leprosy, chronic infection

rapid identification for acid fast + microorganisms

why is acid fast used?

bc some species esp mycobacterium species dont stain w simple/Gram stains bc they have a lot of lipoidal material like mycolic acid

what is mycolic acid?

long chain fatty acids, waxy outer layer (mycomembrane)

cells w mycolic acid are?

acid fast, resistance to antibiotics, can evade immune cells

have a thick hydrophobic cell wall (waxy) and resist aq staining so we use special staining

What is done when conducting the Ziehl-Neelson procedure?

1. smear flooded w carbolfuchsin (dark red, with 5% phenol) - high affinity for lipids in the cell wall

2. smear heated to penetrate dye; retain stain even with decolourizing agent (acid alcohol)

3. counterstain w methelyne blue basic dye

what should we expect to see after staining?

acid fast (microbes w mycolic acid cell walls) = red

non acid fast = blue

What is the Kinyoun modification?

also called the cold stain

concs of phenol + carbolfuchsin are increased and detergent is also added so we dont need to heat

are all acid fast + organisms Gram +?

YES

are all Gram + organisms acid fast +?

NO

exp 6 learning objectives

1. examine species to see if they have mycolic acid in cell wall

2. diff btwn acid fast + / -

3. understand relation btwn acid fast bact and clinical applications and pathogenicity

Acid Fast Technique - method

1. smear is made on agar slant and it will be waxy bc of high mycolic acid lvls in the cell wall (mycobacterium)

2. aseptically prepare heat fixed slides of organisms; take one loopful of M. phlei from agar slant to drop of water on slide. take S. epidermidis from broth tube.

3. air dry + heat fix smears.

4. IN FUMEHOOD, put 4-5 drops of carbolfuchsin on slide, stain for 5-10 mins. keep moist by adding more drops

5. rinse xs with tap water for 30secs

6. drain by blotting corner w absorbent towel.

7. decolorize w acid alc applied dropwise until wash draining from slide is colourless (20-30 secs)

8. rinse w tap water, drain, counterstain w methylene blue for 2min

9. rinse w water, blot dry. examin under oil lens.

Results. -what should we have observed?

Acid Fast + = M. phlei

Acid Fast - = S. epidermidis

Results - Staphylococcus epidermidis

staphylococcus

acid fast -

stained blue, retains methelyne blue

Results - Mycobacterium phlei

bacillus

acid fast +

stained red/pink, retains carbolfuchsin