Question

In Direct Epifluorescent Filter Technique (DEFT) method, staining with acridine orange results in what colour of fluorescence by dead cells? What is the cellular constituent causing this fluorescence?

• A. Orange and RNA
• B. Orange and DNA
• C. Green and RNA
• D. Green and DNA

Hint:

DEFT Acridine Orange Fluorescence:
Dead Inactive Cells = GREEN fluorescence caused by double-stranded DNA.
Live Active Cells = ORANGE-RED fluorescence caused by single-stranded RNA.

Answer 1

The Correct Option is D

Step 1: Understanding the Concept:
Differential epifluorescence microbial viability: Acridine Orange intercalates as a monomer into double-stranded DNA in non-metabolizing/dead cells (green fluorescence at 530 nm) and binds as aggregates to single-stranded ribosomal RNA in active metabolizing cells (orange-red fluorescence at 640 nm).
Key Formula or Approach:
\[ \text{DEFT Acridine Orange Staining: \quad \begin{cases} \text{Active / Living Cells (High Ribosomal RNA)} \& \longrightarrow \mathbf{Orange \text{-Red Fluorescence}} \text{Non-viable / Dead Cells (Double-stranded DNA)} \& \longrightarrow \mathbf{Green \text{ Fluorescence}} \end{cases} \]

Step 2: Detailed Explanation:
In rapid dairy microbiological diagnostics (Direct Epifluorescent Filter Technique - DEFT, Pettipher 1980):
- DEFT is a standardized rapid optical microscopy method for enumerating bacteria in raw milk within 25--30 minutes:
1. Milk is treated with trypsin and Triton X-100 to lyse somatic cells and fat globules, filtered through a $0.6\;\mu\text{m}$ polycarbonate membrane, and stained with the metachromatic fluorochrome Acridine Orange.
2. Dead Inactive Cells: Possess degraded single-stranded RNA; acridine orange intercalates exclusively into intact native double-stranded DNA, emitting Green Fluorescence (D) at $530\text{ nm}$.
3. Live Actively Growing Cells: Possess high cytoplasmic concentrations of single-stranded RNA, causing dye aggregation that emits intense Orange-Red Fluorescence at $640\text{ nm}$.

Step 3: Final Answer:
Hence, dead cells show Green and DNA fluorescence, corresponding to option (D).