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?

<p>It requires a fluorescent microscope (D)</p> Signup and view all the answers

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

<p>Giemsa Stain/Wright Stain (A)</p> Signup and view all the answers

What should be done with specimens soon after collection?

<p>Transported and processed ASAP (B)</p> Signup and view all the answers

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

<p>Pulling the affected hair with sterile forceps (D)</p> Signup and view all the answers

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

<p>Applying 70% alcohol to the area (A)</p> Signup and view all the answers

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

<p>30 ÌŠC (A)</p> Signup and view all the answers

What method is recommended for collecting nail samples?

<p>Cleaning with 70% alcohol and scraping the surface (B)</p> Signup and view all the answers

How long should blood specimens ideally be processed after collection?

<p>8-9 hours (C)</p> Signup and view all the answers

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

<p>Grocott's methenamine silver (GMS) staining (C)</p> Signup and view all the answers

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

<p>Room temperature or 4 ÌŠC (A)</p> Signup and view all the answers

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

<p>Collecting from a catheter bag (A)</p> Signup and view all the answers

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

<p>To dissolve keratin and cellular materials (C)</p> Signup and view all the answers

How should tissue specimens be transported to ensure their integrity?

<p>Transported in sterile saline without fixatives (B)</p> Signup and view all the answers

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

<p>Empty the bladder followed by prostatic massage (D)</p> Signup and view all the answers

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

<p>Bronchoalveolar lavage (C)</p> Signup and view all the answers

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

<p>India ink (A)</p> Signup and view all the answers

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

<p>At 4 ÌŠC for up to 12 hours (D)</p> Signup and view all the answers

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

<p>Direct examination provides immediate results (A)</p> Signup and view all the answers

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

<p>Between 25% and 50% (C)</p> Signup and view all the answers

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

<p>It dislodges fungal structures (C)</p> Signup and view all the answers

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

<p>Periodic acid Schiff stain (D)</p> Signup and view all the answers

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

<p>To clear the specimen for better visibility (C)</p> Signup and view all the answers

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

<p>Methenamine silver stain (C)</p> Signup and view all the answers

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

<p>It can confuse fungal elements with malignant cells (A)</p> Signup and view all the answers

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

<p>Masson-Fontana stain (D)</p> Signup and view all the answers

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

<p>Specialized training method (B)</p> Signup and view all the answers

What is a characteristic feature of Coccidioides immitis?

<p>Sperules may contain endospores (C)</p> Signup and view all the answers

Which organism is known for having hyaline septate hyphae?

<p>Aspergillus spp. (C)</p> Signup and view all the answers

What morphological feature is typically seen in Candida species?

<p>Single budding with pseudohyphae (C)</p> Signup and view all the answers

What type of hyphae is associated with Mucor and Rhizopus?

<p>Large and ribbonlike with occasional septa (A)</p> Signup and view all the answers

What indicates an ectothrix infection in hair?

<p>Arthroconidia on the periphery of the hair shaft (C)</p> Signup and view all the answers

Which fungus is characterized by short, curved hyphal elements?

<p>Malassezia furfur (C)</p> Signup and view all the answers

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

<p>Chains of arthroconidia (B)</p> Signup and view all the answers

What type of hyphae does Geotrichum spp. exhibit?

<p>Rectangular arthroconidia and varying shapes (C)</p> Signup and view all the answers

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

<p>To detect Histoplasma capsulatum and Cryptococcus neoformans (A)</p> Signup and view all the answers

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

<p>Large, double refractile cells (B)</p> Signup and view all the answers

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

<p>To identify Candida albicans (C)</p> Signup and view all the answers

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

<p>Paracoccidiodes brasiliensis (B)</p> Signup and view all the answers

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

<p>They have not been proven effective in clinical settings. (C)</p> Signup and view all the answers

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

<p>Specimens must be fresh to observe elements. (D)</p> Signup and view all the answers

Which test is used to identify Cryptococcus species?

<p>Urease test (B)</p> 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?

<p>Malassezia furfur (A)</p> Signup and view all the answers

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

<p>Sporothrix schenkii (D)</p> Signup and view all the answers

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

<p>35-37°C (C)</p> Signup and view all the answers

Flashcards

Hair Specimen Collection

Pull affected hair with sterile forceps, cutting the hair close to the scalp is less effective. Infected hair fluoresces under a Wood's Lamp. Place samples directly into a sterile Petri dish and incubate on a fungal medium at 25-30°C.

Skin Specimen Collection

Apply 70% alcohol before sampling. Scrape the outer edge of a lesion using a scalpel or glass slide. Culture a portion on a medium and another on a slide with 10% KOH.

Nail Specimen Collection

Submit as scrapings or cuttings, or occasionally as a full nail. Clean with 70% alcohol before scraping. Scrape the surface of the nail and beneath it for excess keratin.

Blood Specimen Collection

Draw blood into a lysis centrifugation system, concentrate the sample, and plant it onto culture media or into biphasic agar/broth bottles. Process within 8-9 hours of collection.

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Blood Culture Technique

Blood is optimal for recovering Histoplasma capsulatum and other filamentous fungi. Requires 10-14 days for H. capsulatum recovery. Optimal temperature for growth is 30°C and incubation time is 21 days.

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Bone Marrow Specimen Collection and Examination

Aspirate from the iliac crest or sternum. Examine the sample under a microscope after Gram staining or GMS staining. This highlights fungal elements and allows visualization of hyphae, conidia, or yeast.

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India Ink Preparation for CSF

A drop of concentrated CSF is prepared with India ink to detect the presence of encapsulated yeast, particularly Cryptococcus neoformans.

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Aspiration of Abscess Fluid

A process where a sterile needle and syringe are used to withdraw fluid from a lesion, such as an abscess, to be plated directly onto media for cultivation.

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First Morning Sputum

The first morning sputum specimen, expectorated through a deep cough, is highly recommended for detecting respiratory infections.

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Prostatic Secretions Collection

Prostatic massage is performed after bladder emptying to obtain prostatic secretions for culture. This involves massaging the prostate gland to expel secretions.

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Direct Microscopic Examination of Vaginal Secretions

A direct microscopic examination of vaginal secretions is often preferred over culture because it provides a quicker and more accurate assessment of the presence of infections.

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KOH Preparation

This technique uses a 10-20% solution of potassium hydroxide (KOH) to dissolve keratin and cellular debris, allowing for visualization of fungal elements in specimens.

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Calcofluor White Staining

This staining method involves the use of calcofluor white, a fluorescent dye that binds to chitin in fungal cell walls, making them easily visible under a fluorescence microscope.

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Methylene Blue Staining

This method uses methylene blue dye to detect Malassezia furfur, the causative agent of tinea versicolor, in skin scrapings and other body fluids.

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Periodic Acid Schiff (PAS) Stain

A staining method that highlights polysaccharides in the cell wall, resulting in a bright red or purplish-red/magenta color.

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Grocott-Gomori Methenamine-Silver Nitrate (GMS) Stain

A staining technique used to visualize fungal elements, which appear black against a green background.

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Giemsa Stain / Wright Stain

A stain often utilized to identify Histoplasma capsulatum and Cryptococcus neoformans in biological samples such as blood and bone marrow. Yeast cells appear purple-blue with a distinct halo.

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Masson-Fontana Stain

A staining method employed to detect melanin in cell walls, which causes fungal elements to appear brown against a pink-purple background.

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Seller's Stain

A staining technique that utilizes Methylene Blue and Basic Fuchsin to effectively stain Cryptococcus neoformans specimens, particularly in brain tissue smears.

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Lactophenol Cotton Blue Wet Mount

A common stain used for observing fungi, employing lactic acid to preserve fungal structures and creating permanent slides.

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Potassium Hydroxide Preparation

A method to clear specimens for quick identification of fungal elements.

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Methenamine Silver Stain

A stain that utilizes silver salts to highlight fungal structures in tissue samples.

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Papanicolaou Stain

A stain primarily used for detecting malignant cells, but can also reveal fungal elements staining pink to blue.

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Periodic Acid Schiff Stain

A staining method that effectively highlights many fungal elements, but struggles to stain certain types of bacteria effectively.

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Cryptococcus neoformans in CSF

A test that may indicate meningitis, appearing positive in less than half of all meningitis cases.

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Cryptococcus neoformans in CSF Sensitivity

A test for Cryptococcus neoformans in cerebrospinal fluid, with lower accuracy in non-HIV infected individuals.

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Saline Wet Mount

A microscopic study of a fungal specimen using a saline solution and a coverslip.

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Wright's Stain

A staining technique used to detect the presence of Histoplasma capsulatum and Cryptococcus neoformans in blood smears or bone marrow.

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Germ Tube Test

A technique used to identify Candida albicans based on the formation of germ tubes when incubated in human serum.

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Urease Test

A test that determines if an organism produces urease, an enzyme found in some fungi, especially within the Cryptococcus genus.

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CHO Assimilation Test

A test that identifies the ability of yeast and yeast-like organisms to utilize different carbon sources for growth.

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Histoplasma capsulatum

Small, round to oval-shaped budding cells often found in clusters within histocytes.

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Sporothrix schenkii

Small, oval to round, sometimes cigar-shaped cells with single or multiple buds.

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Cryptococcus neoformans Complex

Usually spherical, sometimes football-shaped cells with single or multiple buds.

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Malassezia furfur

Small, bottle-shaped cells with buds separated from the parent cell by a septum.

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Blastomyces dermatitidis

Large, double-refractive cells with several buds connected by a broad base.

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Spherules: Coccidioidomycosis

Spherules are round, thick-walled structures containing endospores, often found in coccidioidomycosis. They can be visualized in clinical specimens and are diagnostic of the infection.

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Candida Budding

In Candida infections, yeast cells typically bud off forming a chain-like structure. These constricted structures at the ends resemble links of a sausage.

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Mucorales Hyphae

Mucorales, a group of fungi causing mucormycosis, present as large, ribbon-like hyphae with occasional septa. Their appearance can sometimes be mistaken for Aspergillus.

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Dermatophyte Hyphae

Dermatophytes, fungi causing skin and nail infections, exhibit hyaline septate hyphae and chains of arthroconidia.

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Aspergillus Hyphae

Aspergillus spp. produce septate hyphae that branch dichotomously at a 45-degree angle. They are often disturbed and resemble Zygomycetes.

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Geotrichum spp.

Geotrichum spp. are characterized by the presence of rectangular arthroconidia with a tendency for hyphae and irregular forms.

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Trichosporon spp.

Trichosporon spp. exhibit hyphae, rectangular arthroconidia, and occasionally blastoconidia. These features contribute to its identification in clinical samples.

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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).
  • 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.

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