Microscope, Gram stain, Acid Fast, FITC, Molecular Assay Flashcards
Number of bacteria needed for broth turbidity (visible to naked eye)
10^6 or 1,000,000 bacteria/mL
Light Microscopy Sensitivity
10^5 or 100,000 bacteria/mL
Fluorescent Microscopy Sensitivity
10^4 or 10,000 bacteria/mL
Microscopy where bacteria are seen
Bright-field, Digital, Fluorescence, Phase Contrast, Limited use of Dark Field
Microscopy where fungi are seen
Bright-field, Digital, Fluorescence, Phase Contrast
Microscopy where parasites are seen
Bright-field, Digital, Fluorescence, Phase Contrast, Electron (Limited use)
Microscopy where viruses are seen
Digital, Fluorescence, Electron
Most used smear in bright-field
Fixed smear
Microscopy for aquatic environmental bacteria
Digital Holographic Microscope (Digital Microscopy, 100x greater resolution than bright-field)
Specific detection of microbes by tagging (probes, enzymes)
Fluorescence Microscopy
Visualization of living and non-living cells in unfixed smear/wet smear
Phase Contrast Microscopy
Microscopy only for spirochetes
Dark Field Microscope
Non-conventional microscopy for diagnostic purposes
Electron Microscope
Gram-positive bacteria color
Purple
Gram-negative bacteria color
Pink
Gram staining procedure
Fix with methanol or heat, apply primary stain (10 to 30 seconds), rinse with tap water, apply mordant (twice as long as primary stain), rinse with tap water, apply decolorizer (10 seconds or less), rinse immediately with tap water (longer for thicker smears), apply counterstain (30 seconds), rinse with tap water, blot dry with paper towels, bibulous paper, or air dry (air dry for delicate smears).
Primary stain for Gram stain
Crystal violet
Mordant for Gram stain
Gram’s iodine
Decolorizer for Gram stain
Alcohol and/or acetone
Counterstain for Gram stain
Safranin
Ziehl-Neelsen Acid-fast positive bacilli color
Red (or bright red or pink) against blue or green background
Ziehl-Neelsen Acid-fast negative color
Same as background (blue or green)
Ziehl-Neelsen Primary stain
Carbolfuchsin red
Ziehl-Neelsen Decolorizer
3% HCl in 95% ethanol (HCl, alcohol)
Ziehl-Neelsen Counterstain
Methylene blue or malachite green
Ziehl-Neelsen Procedure
Fix smear (60°C for at least 10 minutes), apply primary stain, heat to almost boiling (should steam), sit for 5 minutes (do not allow to dry out), wash slides with distilled water, apply decolorizer (1 minute), wash with water, apply counterstain (1 minute), wash with distilled water, air dry (do not blot), examine under 400x for screening, confirm suspicious organisms at 1000x magnification using an oil-immersion lens.
FITC Interpretation (no apple-green fluorescence)
Negative
FITC Interpretation (faint yet unequivocal apple-green fluorescence)
1+
FITC Interpretation (apple-green fluorescence)
2+
FITC Interpretation (bright apple-green fluorescence)
3+
FITC Interpretation (brilliant apple-green fluorescence)
4+
Nucleic Acid Hybridization
Filter/Membrane hybridization, Sandwich hybridization, In Situ Hybridization
Filter/Membrane Hybridization Examples
Southern Blot, Northern Blot
In Situ Hybridization Examples
FISH, PNA-FISH
Signal Amplification Hybridization
Branched DNA (bDNA), Hybrid Capture, Cleavase-Invader (Isothermal Cycling Probe Technology)
Nucleic Acid Amplification (PCR-Based)
Conventional/End-point PCR, Real-Time PCR, RT-PCR, Real-Time RT-PCR, Nested PCR, Digital PCR, Multiplex PCR
Nucleic Acid Amplification (Non-PCR-Based)
Nucleic Acid Sequence-Based Amplification (NASBA), Transcription-Mediated Amplification (TMA), Strand Displacement Amplification (SDA)
Nucleic Acid Sequencing
Sanger Sequencing, Pyrosequencing, Next-Generation Sequencing (Massive Parallel Sequencing)
