Specimen Collection and Handling Protocols

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Questions and Answers

What color do polysaccharides stain when using the Periodic Acid Schiff (PAS) stain?

Black on green background

Purple blue with clear halo

Brown on pink-purple background

Bright red or purple red/magenta (correct)

Which stain is effective for detecting fungal elements that appear black on a green background?

Masson-Fontana Stain

Grocott-Gomori Methenamine-Silver Nitrate (GMS) Stain (correct)

Periodic Acid Schiff (PAS) Stain

Giemsa Stain

What is the appearance of Cryptococcus neoformans when stained using Seller’s Stain?

Brown on pink-purple background

Stains with Methylene Blue and Basic Fuchsin (correct)

Purple blue with clear halo

Black on green background

What is a notable disadvantage of the Calcofluor White stain?

It requires a fluorescent microscope Signup and view all the answers

Which stain is used to detect Histoplasma capsulatum in blood and bone marrow samples?

Giemsa Stain/Wright Stain Signup and view all the answers

What should be done with specimens soon after collection?

Transported and processed ASAP Signup and view all the answers

Which method is used to obtain hair samples for fungal analysis?

Pulling the affected hair with sterile forceps Signup and view all the answers

When collecting skin samples, which of these steps should be performed first?

Applying 70% alcohol to the area Signup and view all the answers

For blood samples, what is the optimal temperature for fungal blood cultures?

30 ̊C Signup and view all the answers

What method is recommended for collecting nail samples?

Cleaning with 70% alcohol and scraping the surface Signup and view all the answers

How long should blood specimens ideally be processed after collection?

8-9 hours Signup and view all the answers

Which staining method is commonly used for examining bone marrow samples?

Grocott's methenamine silver (GMS) staining Signup and view all the answers

If hair samples are delayed, what is the recommended storage temperature?

Room temperature or 4 ̊C Signup and view all the answers

Which method is NOT appropriate for collecting a first morning urine specimen?

Collecting from a catheter bag Signup and view all the answers

What is the main purpose of using KOH preparation in direct microscopic examination?

To dissolve keratin and cellular materials Signup and view all the answers

How should tissue specimens be transported to ensure their integrity?

Transported in sterile saline without fixatives Signup and view all the answers

To collect prostatic secretions effectively, one must perform which of the following steps first?

Empty the bladder followed by prostatic massage Signup and view all the answers

What type of respiratory specimen involves introducing saline into the lungs during bronchoscopy?

Bronchoalveolar lavage Signup and view all the answers

Which stain is specifically used in direct microscopic examination of CSF to detect encapsulated yeast?

India ink Signup and view all the answers

What is the recommended storage condition for urine specimens if processing is delayed?

At 4 ̊C for up to 12 hours Signup and view all the answers

Why is direct microscopic examination often preferred over culture for vaginal secretions?

Direct examination provides immediate results Signup and view all the answers

What percentage of meningitis cases show a positive result for neoformans in CSF?

Between 25% and 50% Signup and view all the answers

What is a significant drawback of the lactophenol cotton blue wet mount method?

It dislodges fungal structures Signup and view all the answers

Which of the following stains effectively detects fungal elements but does not stain Nocardia spp. well?

Periodic acid Schiff stain Signup and view all the answers

What is the primary function of potassium hydroxide in the preparation of fungal specimens?

To clear the specimen for better visibility Signup and view all the answers

Which staining method is best suited for detecting fungal elements in histologic sections?

Methenamine silver stain Signup and view all the answers

What is a disadvantage of the Papanicolaou stain in the examination of secretions?

It can confuse fungal elements with malignant cells Signup and view all the answers

Which stain is specifically helpful in differentiating melanin and hemosiderin pigments?

Masson-Fontana stain Signup and view all the answers

What type of training is required for effectively using the Methenamine silver stain?

Specialized training method Signup and view all the answers

What is a characteristic feature of Coccidioides immitis?

Sperules may contain endospores Signup and view all the answers

Which organism is known for having hyaline septate hyphae?

Aspergillus spp. Signup and view all the answers

What morphological feature is typically seen in Candida species?

Single budding with pseudohyphae Signup and view all the answers

What type of hyphae is associated with Mucor and Rhizopus?

Large and ribbonlike with occasional septa Signup and view all the answers

What indicates an ectothrix infection in hair?

Arthroconidia on the periphery of the hair shaft Signup and view all the answers

Which fungus is characterized by short, curved hyphal elements?

Malassezia furfur Signup and view all the answers

What is a distinguishing feature of dermatophytes in nail and skin samples?

Chains of arthroconidia Signup and view all the answers

What type of hyphae does Geotrichum spp. exhibit?

Rectangular arthroconidia and varying shapes Signup and view all the answers

What is the primary use of Wright's stain in clinical settings?

To detect Histoplasma capsulatum and Cryptococcus neoformans Signup and view all the answers

Which of the following is NOT a characteristic feature of Histoplasma capsulatum?

Large, double refractile cells Signup and view all the answers

What is the function of the germ tube test in clinical mycology?

To identify Candida albicans Signup and view all the answers

Which organism is characterized by cells that appear with a 'Mariner’s Wheel' appearance?

Paracoccidiodes brasiliensis Signup and view all the answers

Why has molecular detection methods not been accepted as a routine diagnostic tool in clinical mycology?

They have not been proven effective in clinical settings. Signup and view all the answers

What is a key limitation of the saline wet mount preparation?

Specimens must be fresh to observe elements. Signup and view all the answers

Which test is used to identify Cryptococcus species?

Urease test Signup and view all the answers

Which of the following fungi is described as small, bottle-shaped cells with buds separated from the parent cell by a septum?

Malassezia furfur Signup and view all the answers

Which fungus typically exhibits oval to round, cigar-shaped forms with single or multiple buds?

Sporothrix schenkii Signup and view all the answers

What is the ideal incubation temperature for conducting a germ tube test?

35-37°C Signup and view all the answers

Study Notes

Specimen Collection, Handling, and Transport

Specimens should be transported and processed as quickly as possible (ASAP).
Generally, specimens should not be frozen or allowed to dry.
Store specimens at room temperature (25°C to 30°C).

Specific Types and Recommended Protocols

Abscess fluids/wound exudates
Respiratory specimens
Urine
Prostatic secretions
Vaginal secretions
Stool
Blood
Bone marrow
CSF
Tissue
Other sterile body fluids
Hair
Skin
Nails

Hair

Pull affected hair with sterile forceps; cutting the hair close to the scalp is less effective.
Infected hair fluoresces when viewed under a Wood's Lamp.
Place samples directly into a sterile petri dish.
Incubate a few pieces on a fungal medium at 25-30°C.
Store at room temperature or 4°C if there is a delay.

Skin

Apply 70% alcohol before sampling.
Scrape samples from the outer edge of a surface lesion with a scalpel or glass slide.
Place a portion of the sample on a slide with 10% KOH; inoculate another portion onto the culture medium.
Store at room temperature.

Nails

Submit nails as scrapings, cuttings, or sometimes as complete nails.
Clean nails with 70% alcohol before scraping.
Scrape the nail surface before sampling.
Scrapings of excess keratin beneath the nail are an alternative.

Blood

Draw blood into a lysis centrifugation system.
Plant the concentrate onto culture media.
Biphasic Agar/Broth Bottles are available.
Process specimens within 8-9 hours of collection.
Optimal isolation for Histoplasma capsulatum and other filamentous fungi.
10–14 days for H. capsulatum recovery.
Suggested incubation time is 21 days at 30°C.

Bone Marrow

Typically collected through aspiration from the iliac crest or sternum.
Examine the sample under a microscope after Gram staining or Grocott's methenamine silver (GMS) staining. (GMS highlights fungal elements).
Direct visualization of fungal cells (hyphae, conidia, or yeast) helps indicate infection.

CSF and Other Sterile Body Fluids

Concentrate specimens before inoculation.
Use a drop of concentrate for India Ink preparation.
For large volumes (>5 mL), filter through a membrane filter (0.45 μm) and place onto media.
Store specimens at 30°C or room temperature if processing is delayed.

Abscess Fluid/Wound Exudates

Disinfect the skin over the lesions.
Aspirate fluid using a sterile needle and syringe.
Plate fluid directly onto media.

Respiratory Specimens

(A) Bronchial brushings
(B) Bronchoalveolar lavage (BAL): Saline solution is introduced into the lungs during bronchoscopy and retrieved.
(C) Transtracheal aspirates: A needle is inserted through the skin into the trachea to collect secretions directly from the respiratory tract.
(D) Sputum: First early morning specimen, expectorated through a deep cough.
(E) Upper respiratory tract specimens: Direct examination is important. Curettage samples and swabs are acceptable.

Urine

First morning urine is preferred, but a random sample is also acceptable.
24-hour collections and catheter bag samples are not acceptable.
Store at 4°C for up to 12 hours.

Prostatic Secretions

Empty the bladder, then perform a prostatic massage.
Inoculate secretions directly onto appropriate culture media.

Vaginal Secretions

Direct microscopic examination of secretions is preferable to culture.
Cultures can sometimes be misleading.
Swabs are suitable.
Store at 4°C.

Stool

Cultures can sometimes be misleading.
Biopsy is preferred.
Store at 4°C.

Tissue

Transport in sterile saline without fixatives.
Avoid freezing or dehydration.
Mince into 1 mm cubes before inoculation, grinding, or stomached.
Store at room temperature if processing is delayed.

Methods of Identifying Fungi

Direct Microscopic Examination of Specimens
- Wet Mounts:
  - KOH preparation (10-20% KOH): Dissolves keratin and other cellular materials.
  - KOH with Calcofluor White: Calcofluor binds to polysaccharides in chitin or cellulose.
  - Methylen Blue: Detects tinea versicolor from skin scrapings and other body fluids.
  - India Ink (China Ink): Detects encapsulated yeasts (Cryptococcus neoformans) in CSF. -Tissue Stains:
  - Periodic Acid Schiff (PAS): Attaches to polysaccharides in cell walls (red/magenta). Grocott-Gomori Methenamine Silver Nitrate (GMS): Highlights fungal elements (black on green). Giemsa Stain/Wright Stain: Detects Histoplasma capsulatum and Cryptococcus neoformans (purple-blue with clear halo in blood/bone marrow). Masson-Fontana Stain: Stains melanin in cell walls, fungus appears brown on pinkish-purple background. Seller's Stain: Effective for staining Cryptococcus neoformans in brain tissue smears.

Molecular Detection Methods

Increasing use in clinical microbiology.
Not yet a routine diagnostic tool in the field of clinical mycology.

Additional Tests

Germ tube test: Identifying Candida albicans.
Urease test: Identifying Cryptococcus organisms.
CHO assimilation test: Identifying yeast and yeast-like organisms.

Germ Tube Test Procedure

Inoculate a pure culture of Candida into 0.5 mL of human serum.
Incubate at 35–37°C for 2–3 hours.
Remove 1 or 2 drops of yeast cells and place on a slide.
A positive result is the production of a germ tube.

Summary of Characteristic Features of Fungi (in Direct Examination)

Provides descriptions of various fungal forms and features.

Summary of Methods for Direct Microscopic Detection of Fungi

Provides details on different methods for detecting fungi.

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Description

This quiz covers essential protocols for the collection, handling, and transport of various biological specimens. It includes specific procedures for different types of specimens, such as hair, skin, and fluids, emphasizing safe and effective transportation methods.