Acid-Fast staining + Durham glucose tube + Kligler iron test

Acid-fast is also known as

Ziehl-Nelson method

Purpose of Acid-fast staining

to differentiate acid-fast from non-acid-fast bacteria based on cell walls

What bacteria is and is not acid-fast

acid-fast - Mycobacterium smegatis

Non-acid-fast - staphlyococcus epidermidis

Steps of Ziehl-Nelson method

1. Primary stain with Basic fuchsin - stains acid-fast bacteria red/pink

2. Decolorizer - Acid-alcohol - removes primary stain from non-acid-fast bacteria (staphylococcus epidermidis)

   1. Counterstain - Methylene blue - stains non-acid-fast bacteria blue

How is the decolorizer different in acid-fast than gram-staining

Acid-fast uses acid-alcohol while gram staining uses alcohol

Why do we steam the smear

Heat acts a mordant and helps primary stain penetrate the waxy cell wall

Why is the cell wall waxy

Mycolic acid is waxy lipid in the cell wall of acid-fast bacteria that makes wall resistant to ordinary staining

• helps bacteria retain the primary stain

Durham glucose tube

test to see if bacteria can ferment glucose to produce acid and gas

• acid production and gas production are separate reactions

Results of Durham test

Broth color Durham tube results

Yellow Bubble +/+
Yellow No bubble +/-
Red Bubble -/+
Red No Bubble -/-

• Yellow = acid = positive fermentation

• red = no acid detected = negative fermentation

• Bubble = gas = positive gas production

• No babble = no gas = no gas production

Kligler’s iron Agar (KIA) tube

helps determine whether bacteria ferment glucose and lactose

• either aerobically or whether they produce H2S (anaerobically)

2 parts of tube

Slant - angled surface exposed to oxygen, where aerobic metabolism can occur

• indicator for lactose production

Butt - deeper bottom, where oxygen is limited and fermentation occurs

• indicator for glucose production

Results of KIA test

Slant/Butt Interpretation

Red/Yellow (K/A) Glucose +, lactose -

Y/Y (A/A) glucose +, lactose +

R/R (K/) glucose -, lactose -

Black Precipitate = H2S positive (+)

• H2S reacts with iron producing black precipitate

• Yellow means glucose (+)

• Red slant ( lactose -)

• Yellow slant (lactose +)

K = Alkaline (red) - agar is basic

A = Acid (yellow) agar is acidic due to sugar fermentation