MLS 334 EXAM 2 STUDY GUIDE
Explain the importance of direct examination in the microbiology lab - Answer -It helps
guide physicians for empiric treatment and helps develop epidemiologic data
Direct examination: Specimen Processing - Answer -Ensure specimen is properly
labeled (2 identifiers, specimen type, date of collection, time of collection, collectors'
initials)
Slide Prep: Swab, Liquid, and Colony from plate - Answer -Swab:
Roll across slide evenly, dab tip of swap on slide
Liquid:
Use a sterile tool (pipette, loop, swab) and put a thin smear of liquid onto the slide
Colony from plate:
Place a drop of deionized water onto the slide. Take 1 well isolated colony from the
plate using a clean loop and smear into DI water
Cytocentrifuge - Answer -Cellular elements and microorganisms arranged in a
monolayer on slide
Simple stains - Answer -Looking at color and shapes.
Example: Wright-Giemsa
Differential stains - Answer -Looking at color specific components
Example: Gram, acid fast, calcofluor white
Probe mediated stains - Answer -Looking at specific organism ID
Example: antibody or DNA probe stains
Purpose and principle of the gram stain - Answer -Bacteria will retain stain based on
properties of their cell wall. Gram-positive will show purple/blue because they retain
crystal violet. Gram-negative will show pink/red because they retain safranin.
Gram stain Procedure - Answer -1. Flood the slide with crystal violet for 30 seconds
2. Rinse with water
3. Flood the slide with iodine for 30 seconds
4. Rinse with water
5. Decolorize using the provided decolorizer
6. Rinse with water immediately
7. Flood the slide with safranin for 30 seconds
8. Rinse with water
, 9. Allow slide to dry
What is the appropriate grading and interpretation for sputum samples? - Answer -On
10X quantitate squamous epithelial cells and WBC. Reject sputum sample if there are
>25 SEC present and any amount of WBC. If acceptable, then continue onto 100x to
quantitate bacteria.
Quality Gram stain: - Answer -Nice pink background
No stain precipitate
Can visually see whether gram +/- and cocci/rods
On sputum: No epithelial cells present
Poor Gram Stain: - Answer -Can't even tell what your looking at
Crystal violet precipitate
Too Thick of a stain
Over decolorized
Acid-Fast Stain: Purpose, Principle, & Interpretation - Answer -Purpose
differential stain to identify acid-fast organisms
Principle:
carbolfuchsin can penetrate the cell wall
Interpretation:
Acid-fast cells will stain fuchsia
Non-acid fast cells will stain blue
KOH: Purpose, Principle, and Procedure - Answer -Purpose:
Detecting fungal elements in skin, hair, nails, and tissue
Principle:
KOH will break down keratin and skin for easier interpretation
Procedure:
1 drop 10-20% KOH is added to a slide
Add specimen to drop and coverslip
Heat slide and cool
Read microscopically
Calcofluor White: Purpose, Principle, Procedure, and Interpretation - Answer -Purpose:
detecting fungal elements in skin, hair, nails, and tissue
Principle:
Calcofluor white will bind to polysaccharides present in the chitin cell wall of the fungus
Procedure:
Explain the importance of direct examination in the microbiology lab - Answer -It helps
guide physicians for empiric treatment and helps develop epidemiologic data
Direct examination: Specimen Processing - Answer -Ensure specimen is properly
labeled (2 identifiers, specimen type, date of collection, time of collection, collectors'
initials)
Slide Prep: Swab, Liquid, and Colony from plate - Answer -Swab:
Roll across slide evenly, dab tip of swap on slide
Liquid:
Use a sterile tool (pipette, loop, swab) and put a thin smear of liquid onto the slide
Colony from plate:
Place a drop of deionized water onto the slide. Take 1 well isolated colony from the
plate using a clean loop and smear into DI water
Cytocentrifuge - Answer -Cellular elements and microorganisms arranged in a
monolayer on slide
Simple stains - Answer -Looking at color and shapes.
Example: Wright-Giemsa
Differential stains - Answer -Looking at color specific components
Example: Gram, acid fast, calcofluor white
Probe mediated stains - Answer -Looking at specific organism ID
Example: antibody or DNA probe stains
Purpose and principle of the gram stain - Answer -Bacteria will retain stain based on
properties of their cell wall. Gram-positive will show purple/blue because they retain
crystal violet. Gram-negative will show pink/red because they retain safranin.
Gram stain Procedure - Answer -1. Flood the slide with crystal violet for 30 seconds
2. Rinse with water
3. Flood the slide with iodine for 30 seconds
4. Rinse with water
5. Decolorize using the provided decolorizer
6. Rinse with water immediately
7. Flood the slide with safranin for 30 seconds
8. Rinse with water
, 9. Allow slide to dry
What is the appropriate grading and interpretation for sputum samples? - Answer -On
10X quantitate squamous epithelial cells and WBC. Reject sputum sample if there are
>25 SEC present and any amount of WBC. If acceptable, then continue onto 100x to
quantitate bacteria.
Quality Gram stain: - Answer -Nice pink background
No stain precipitate
Can visually see whether gram +/- and cocci/rods
On sputum: No epithelial cells present
Poor Gram Stain: - Answer -Can't even tell what your looking at
Crystal violet precipitate
Too Thick of a stain
Over decolorized
Acid-Fast Stain: Purpose, Principle, & Interpretation - Answer -Purpose
differential stain to identify acid-fast organisms
Principle:
carbolfuchsin can penetrate the cell wall
Interpretation:
Acid-fast cells will stain fuchsia
Non-acid fast cells will stain blue
KOH: Purpose, Principle, and Procedure - Answer -Purpose:
Detecting fungal elements in skin, hair, nails, and tissue
Principle:
KOH will break down keratin and skin for easier interpretation
Procedure:
1 drop 10-20% KOH is added to a slide
Add specimen to drop and coverslip
Heat slide and cool
Read microscopically
Calcofluor White: Purpose, Principle, Procedure, and Interpretation - Answer -Purpose:
detecting fungal elements in skin, hair, nails, and tissue
Principle:
Calcofluor white will bind to polysaccharides present in the chitin cell wall of the fungus
Procedure:
