[2S] UNIT 5 Exercise 5 Flashcards
Have been regarded the foundation of a repertoire of diagnostic tests for detection of fungal infections.
Direct Microscopy
Culture
Histopathology
This does not only enhance visualization of cell components but also provides contrast and can highlight a metabolic process.
Fungal-specific stains
Provides rapid presumptive test for detection of fungal elements such as hyphae and budding yeast, in clinical specimens
Microscopy
Staining of fungal components for light microscopy plays a vital role in diagnosis of encapsulated fungi such as _______, ________ and other medically important fungi.
C. neoformans, Candida spp.
ODD ONE OUT: FACTORS THAT INFLUENCE THE SENSITIVITY OF A MICROSCOPIC EXAMINATION
Individual fungal agent
Source
Quality & Prep of the Specimen
Exp & Expertise of the Lab Scientist
wla
T/F: Factors such as the individual fungal agent, the source, quality and preparation of the specimen, and the experience and expertise of the laboratory scientist influence the sensitivity of a microscopic examination
T
These procedures may require the use of ______ of deep tissues, which may place one at risk particularly those patients who are most susceptible to invasive disease.
biopsies
Due consideration has to be made in the use of direct microscopy and histopathology in the diagnosis of ______ fungal infection
invasive
1st step in collecting skin scrapings & nail clippings
Sterilize the affected skin & nail with 70% alcohol
Skin scrapings are collected by scraping from the ______ _____ of lesion (as edge has greatest amount of viable fungus) with a blunt scalpel blade held at right angles to the skin.
outer edges
T/F: Blunt scalpel blade held at right angles to the skin should be used when collecting skin scrapings
T
Where to place the collected skin scales?
Sterile petri dish / wide-mouthed container
Clean paper w/ adhesive tape and label
Preferred area of the nail clipping collection
Damaged, discoloured, brittle or
dystrophic area
If a skin scraping does not yield sufficient material, then a ____ or ______ _____ could be pressed on the lesion
swab or adhesive tape
T/F: The entire thickness of the damaged nail should be cut as far back as possible. If crumbly material or material under the nail is present, collect also at this site and place in a sterile container.
T
T/F: If skin lesions are present near the nail, carefully scrape the affected area and the collected material should be sent separately.
T
T/F: Since fungal cells and other fungal elements are larger compared to bacteria, the use of dried stained smears prepared from clinical specimen is necessary.
F; unecessary
Very simple and easiest method of examining fungal structures
Wet mount or wet preparation
Routinely used solutions for mounting fluid for microscopic study of fungi
SaPoL
Saline
Potassium hydroxide
Lactophenol cotton blue
PREPARING A WET MOUNT
T/F: Place your specimen on the slide. Make sure the layer is as thick as possible.
F; thin
PREPARING A WET MOUNT
T/F: Overlay the coverslip by gently placing it at one edge of the KOH/Calcoflour white solution and slowly lower it over the sample.
T
PREPARING A WET MOUNT
Add one drop of ___ or _________ _____ (or stain) to the specimen
KOH or Calcoflour white
PREPARING A WET MOUNT
Remove the trapped bubbles by gently pressing the coverslip with the ________ or a blunt _______
eraser end of a pencil or a blunt forcep
Principle: Skin scrapings and nail clipping samples when mixed and suspended in 10-20% respectively will cause the keratin and other proteinaceous components of tissue to dissolve making the fungal elements more visible and prominent
KOH Preparation
SKIN SCRAPINGS - KOH PREP
Percentage of KOH added to the slide
a drop of 10% KOH
SKIN SCRAPINGS - KOH PREP
Leave the slide at room temperature for __ minutes
10
SKIN SCRAPINGS - KOH PREP
T/F: Excess fluid on the slide can be blot off using a piece of tissue paper
T
NAIL CLIPPINGS - KOH PREP
T/F: The nail should be cut into small pieces with the nail cutter
F; scalpel or sterile blade
NAIL CLIPPINGS - KOH PREP
Percentage of KOH added over pieces of nail samples
a drop of 20% KOH
KOH PREP
T/F: Coverslips should be added to the nail clippings & skin scrapings before adding KOH
F; after adding KOH
NAIL CLIPPINGS - KOH PREP
Leave the slide at room temperature for __ minutes
10
Principle: Dermatological samples e.g skin scrapings and nail clipping samples when mixed and suspended in calcofluor white, cause the stain to bind to chitin and cellulose component of fungal cell wall and other fungal elements producing a chalk-white or brilliant apple green fluorescence depending on the filters used
Calcofluor White - KOH Stain Preparation
A very rapid and sensitive method for microscopic observation of fungal structure in dermatological samples and other clinical specimens. This method requires the use of fluorescence microscope fitted with filters that will provide an excitation with ultraviolet light below 400 nm wavelength.
Calcofluor White - KOH Stain Preparation
T/F: Calcofluor White - KOH Stain Preparation requires the use of fluorescence microscope fitted with filters that will provide an excitation with ultraviolet light below 450 nm wavelength.
F; 400 nm
Causes the stain to bind to chitin and cellulose component of fungal cell wall and other fungal elements producing a chalk-white or brilliant apple green fluorescence depending on the filters used
Calcofluor White
CALCOFLUOR WHITE - KOH STAIN PREPARATION
T/F: On a clean microscope slide, add 1 drop of Calcofluor white stain and 1 drop of 10% KOH. Do not mix the two solutions.
F; mix the solution
CALCOFLUOR WHITE - KOH STAIN PREPARATION
On a clean microscope slide, how many drops of calcofluor white stain and 10% KOH should be added?
both 1
CALCOFLUOR WHITE - KOH STAIN PREPARATION
- Cover with a coverslip. Preparation may be squased using the ______ of the inoculation _____ and then blot off the excess fluid.
butt; needle
CALCOFLUOR WHITE - KOH STAIN PREPARATION
Smear from ___________ can be used as positive control
Aspergillus spp.
CALCOFLUOR WHITE - KOH STAIN PREPARATION
T/F: It is recommended that the specimen be stained together with a positive and negative control.
T
CALCOFLUOR WHITE - KOH STAIN PREPARATION
Leave the slide at ____ _________ for _ minutes or gently warm the slide by passing it over a flame or warmer.
room temperature; 5
CALCOFLUOR WHITE - KOH STAIN PREPARATION
T/F: Examine slide for presence of fungal structures (e.g., yeast cells, hyphal structures) under fluorescent microscope following the manufacturer’s recommendation for the required wavelength and filter
T
CALCOFLUOR WHITE - KOH STAIN PREPARATION
Smears from _______ can be used as negative control
E. coli
CALCOFLUOR WHITE - KOH STAIN PREPARATION
T/F: The control smears are stained in the same manner as the specimen.
T
LACTOPHENOL COTTON BLUE LPCB STAIN PREPARATION
Is both a dye and a mounting fluid
Lactophenol cotton blue
LACTOPHENOL COTTON BLUE LPCB STAIN PREPARATION
Preserves the fungal structures
Lactic acid
LACTOPHENOL COTTON BLUE LPCB STAIN PREPARATION
A mixture of phenol that acts as a disinfectant
Lactophenol
LACTOPHENOL COTTON BLUE LPCB STAIN PREPARATION
Inhibits the cellulolytic activity of the fungus
Glycerol
LACTOPHENOL COTTON BLUE LPCB STAIN PREPARATION
An aniline dye that stains the chitin and cellulose of fungal cell wall blue
Cotton Blue
Principle: Lactophenol cotton blue is both a dye and a mounting fluid. Lactophenol is a mixture of phenol that acts as a disinfectant, lactic acid that preserves the fungal structures and glycerol that inhibits the cellulolytic activity of the fungus. Cotton blue is an aniline dye that stains the chitin and cellulose of fungal cell wall blue.
Lactophenol Cotton Blue Stain Preparation
T/F: Nail polish or paramount is a material for KOH preparation
F; for LPCB Wet Mount
TEASE MOUNT METHOD
- Clean the microscopic slide with ____ alcohol. Make sure it is grease or oil-free.
70%
TEASE MOUNT METHOD
Using aseptic technique, sterilize the inoculating needle then allow it to cool. Take a fragment of fungal colony (approximately _____ from the periphery) with a wooden stick or dissecting needle and place it over the LPCB solution.
1-2 mm
TEASE MOUNT METHOD
- Add a small drop of ________ in the middle of the slide.
lactophenol Cotton Blue
TEASE MOUNT METHOD
T/F: 4. Using two wooden sticks, gently tease the fungal element until it has been separated and is thickly spread out in the lactophenol.
F; thinly
TEASE MOUNT METHOD
T/F: 6. Slowly lower the coverslip gently onto the slide. Tap or push the coverslip down to dislodge conidia from conidiophores.
F; Do not tap or push the coverslip down as this may dislodge conidia from conidiophores.
TEASE MOUNT METHOD
- Take a clean coverslip, hold it between your ________ finger and ______ and touch one edge of the mixture with the edge of the coverslip.
index finger and thumb
TEASE MOUNT METHOD
Objectives of the microscope used
Low (x10)
High (x40)
TEASE MOUNT METHOD
- Using aseptic technique, sterilize the inoculating needle then allow it to cool. Take a fragment of fungal colony (approximately 1-2 mm from the periphery) with a _____________ and place it over the LPCB solution.
wooden stick or dissecting needle
TEASE MOUNT METHOD
- Seal the edges of the coverslip using?
nail polish or paramount
A fast, easy to perform and inexpensive method for microscopic examination of fungal structures.
Adhesive Tape Method
It preserves the conidial arrangements of the more delicate filamentous molds and allows one to make an accurate identification
Adhesive Tape Method
ADHESIVE TAPE METHOD
How many times will you add a drop of lactophenol cotton blue?
2 (steps 2 & 8)
ADHESIVE TAPE METHOD
- Open the fungal culture plate with the opposite hand and touch the sticky part of the tape to the ______ of fungal colony (usually on the mycelial part)
surface
ADHESIVE TAPE METHOD
Possible control samples to use
Aspergillus niger or Trichophyton mentagrophytes
COLLECTION OF SKIN SCRAPINGS
Ayusin mo
A. Skin scrapings are collected by scraping from the outer edges of lesion (as edge has greatest amount of viable fungus) with a blunt scalpel blade held at right angles to the skin.
B. Sterilize the affected skin by cleaning the area with 70% alcohol.
C. If a skin scraping does not yield sufficient material, then a swab or adhesive tape could be pressed on the lesion.
D. Place the collected skin scales in a sterile petri dish or similar wide-mouthed container. Skin scrapings may also be collected in a clean, dry piece of paper folded securely with adhesive tape and label it properly.
- B
- A
- D
- C
COLLECTION OF NAIL CLIPPINGS
Ayusin mo
A.Clean the nail with 70% alcohol.
B. Examine the nail for damaged, discoloured, brittle or dystrophic area and collect the material from this site.
C. The entire thickness of the damaged nail should be cut as far back as possible. If crumbly material or material under the nail is present, collect also at this site and place in a sterile container.
D. If skin lesions are present, carefully scrape the affected area and the collected material should be sent separately.
- A
- B
- C
- D
SKIN SCRAPINGS - KOH
Ayusin mo
A. Leave the slide at room temperature for 10 minutes
B. Add a drop of 10% KOH. Apply coverslip.
C. Apply slight pressure over the specimen by gently
pressing the coverslip in order to spread the specimen evenly on the slide. Excess fluid can be blot off using a piece of tissue paper
D. Examine the prepared KOH wet mount under the microscope and look for fungal elements (e.g., yeast cells, hyphal structures)
E. Place skin scraping on slide.
- E
- B
- C
- A
- D
CALCOFLUOR WHITE - KOH STAIN PREPARATION
A. Place the specimen in the mixture.
B. Examine slide for presence of fungal structures (e.g., yeast cells, hyphal structures) under fluorescent microscope following the manufacturer’s recommendation for the required wavelength and filter
C. Leave the slide at room temperature for 5 minutes or gently warm the slide by passing it over a flame or warmer.
D. Cover with a coverslip. Preparation may be squased using the butt of the inoculation needle and then blot off the excess fluid.
E. On a clean microscope slide, add 1 drop of Calcofluor white stain and 1 drop of 10% KOH. Mix the two solutions.
- E
- A
- D
- C
- B
NAIL CLIPPINGS - KOH
Ayusin mo
A. If the specimen is a nail clipping, Cut the nail into small pieces with the scalpel or sterile blade.
B. Examine the prepared KOH wet mount under the microscope and look for fungal elements (e.g. yeast cells, hyphal structures)
C. Add a drop of 20% KOH over pieces of nail samples. Apply coverslip.
D. Gently warm the slide by passing it over a flame or warmer.
E. Gently press the coverslip to spread the softened nail sample evenly on the slide.
F. Leave the slide at room temperature for 10 minutes.
- A
- C
- D
- E
- F
- B
