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Specimen collection in mycology

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Meenakshi Muthuswamy

Fungal specimens collection hair shaft, urine , body fluids, eye swabs, nails, sputum. specific methodology for staining.

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COLLECTION OF SPECIMENS IN MYCOLOGY
COLLECTION OF SPECIMEN GENERAL PRINCIPLE The most important steps for the isolation of fungus are: • proper collection of specimens. • rapid transport to the lab. • prompt and correct processing of the specimen. • inoculation onto appropriate media. • incubation at suitable temperatures. Each specimen submitted must be examined for quality and the importance of proper collection can not be overemphasized. Fungi normally live a saprophytic existence in nature, capable of a life cycle without a human or animal host. Humans become an accidental host by inhalation of spores or by introduction of the fungus into tissue through trauma. Generally, humans are relatively resistant to these infections, but due to long- term catheterization, GI surgical procedures, chemotherapy and alterations of the immune system, exposure of fungi normally considered to be nonpathogenic can lead to infection. .
MICROSCOPIC EXAMINATION: • A. Specimens of any clinical nature can be examined microscopically for fungi upon the physician's request or at the initiative of the laboratorian. • All specimens of sufficient quantity submitted for fungal culture should be examined microscopically for fungi. • When there is not a sufficient quantity of a specimen for both a culture and direct exam, the culture takes priority over the smear because it is more sensitive than microscopic examination. • Observing a fungus in a clinical early information that may be crucial for determining appropriate therapy for the patient.
MICROSCOPIC EXAMINATION: Fungus stains are determined by the type of specimen. • India ink - CSF • KOH wet prep - Hair, nails, and skin scrapings • Gram stain - All other specimens, but a fungus stain performed by the histology section of the laboratory yields better results, particularly for tissue specimens.
MICROSCOPIC EXAMINATION OF INDIA INK PREPARATION
INDIA INK PREPARATION It is usually employed to detect the presence of microorganisms, particularly yeast, in a body fluid. It induces negative staining by creating a black, semi-opaque background that makes the capsule of the yeast clearly visible. One drop of cerebrospinal fluid (CSF) is mixed with a drop of India Ink on a clean micro slide. A cover slip is placed over it and the sample is examined through the low power lens, and then through the high power lens, of a microscope. The preparation can be diluted with a small drop of water if it is too dark and murky.
KOH & FUNGUS CULTURE COLLECTION PROCEDURE • Hair • Scrape the scalp with a blunt scalpel. (Hair stubs, contents of plugged follicles, and skin scales are also acceptable) • Hair may also be plucked from the scalp with forceps. (Do not submit cut hair.) • Place in a dry sterile container. • Transport immediately to the laboratory at room temperature. • Nails • Cleanse the nail with alcohol. • Remove the outermost layer by scraping with a scalpel. • Clippings from any discolored or brittle parts of the nail may be used. • Deeper scrapings and debris under the edges of the nail are also suitable. • Place the specimens in a dry sterile container. • Transport immediately to the laboratory at room temperature. • Skin • Cleanse the skin with alcohol. • Collect epidermal scales with a scalpel at the active border of the lesion. • Place the specimens in a dry sterile container. • Transport immediately to the laboratory at room temperature.
FUNGUS CULTURE COLLECTION PROCEDURE SCALP AND HAIR • Scrape the scalp with a blunt scalpel. • Collect the basal portion of the infected hair. • Note: Infected hairs may be selected by placing the patient under a UV light (Wood’s lamp). • Hairs infected with some dermatophytes will fluoresce under UV light. • Hairs that are fluorescent, distorted or fractured should be cultured. • Place hair in a dry sterile container. • Transport to the laboratory at room temperature. • The following specimens are also acceptable: 1. Hair stubs 2. Contents of plugged follicles 3. Skin scales 4. Hair plucked from the scalp with forceps
FUNGUS CULTURE COLLECTION PROCEDURE VAGINAL SWABS • 1. Using several sterile swabs, collect discharge from the vagina and place swabs in a sterile tube that can be capped. • This method of collection provides a specimen that is also suitable for performance of a wet prep. • 2. Alternatively, Vaginal material may be collected and submitted on a culturette SKIN AND INTERSPACES • 1. Wipe lesions and interspaces between toes with an alcohol sponge. • 2. Scrape the entire lesion(s) and both sides of the interspaces with a sterile scalpel blade. • 3. Place all scrapings into a sterile urine culture cup
FUNGUS CULTURE COLLECTION PROCEDURE TISSUE • 1. Biopsy aseptically from both the center and edge of the lesion. • 2. Place tissue in a sterile container and if transport is delayed, it may be covered with a minimum of sterile saline to prevent drying. RESPIRATORY SPECIMENS OTHER THAN SPUTUM • 1. These include bronchial washings, lung biopsies, and tracheal aspirates. • 2. These specimens are collected aseptically by the physician, placed in the appropriate container and immediately transported to the lab
FUNGUS CULTURE COLLECTION PROCEDURE OTHER SOURCES (Submit fluid or culture swab) • 1. Bone Marrow – collect 0.5 ml aspirated material • 2. Blood – collect 2 sets of blood cultures (40 ml of blood) • 3. Corneal scraping, intraocular aspiration, or biopsy obtained by an ophthalmologist. • 4. Sputum – collect first early morning deep cough sample after patient’s teeth are brushed and the mouth is well rinsed (24 hr. specimens are not satisfactory). • 5. Subcutaneous Specimens (wound, abscess, lesion, pus, drainage) - For suppurative lesions of the deep skin and subcutaneous tissue, where pus may be loculated within abscess or is exuding from deep sinus tracts, aspiration with a sterile needle and syringe should be attempted. The material should be placed in an anaerobic transport. In addition to a fungus culture, both anaerobic and aerobic cultures should be performed. The former being necessary to recover the anaerobic branching filamentous bacteria belonging to the genus Actinomyces. • 6. Tissue – obtained in surgery. Place on sterile 4x4 gauze or in sterile container and transport to the laboratory immediately. • 7. Body Fluids and Exudates – samples are usually obtained by aspiration with a sterile needle and syringe. • 8. Urine – collect first morning “mid-stream” specimen. Do not obtain urine samples from a collection bag or bedpan. • 9. Stool – rarely worth culturing – growth of a large amount of yeast has possible significance, but only in indicating a lack of normal flora.
TYPES OF SPECIMENS… Body fluids (pleural, synovial, peritoneal) • 1. Cleanse proposed puncture site with alcohol, followed by iodine solution. • 2. Using needle and syringe, aspirate aseptically and place in sterile container. Abscess, pus, exudate, drainage 1. Using needle and syringe, aspirate specimen and place in sterile container. 2. 2. Small volume specimens may be submitted in the syringe, without the needle. Recap the syringe with the syringe cap.
TYPES OF SPECIMENS… Eye Swabs • 1. Notify the lab before beginning procedure. • Media will be provided by laboratory to be inoculated at the bedside. 2. For suspected mycotic keratitis, an ophthalmologist will scrape the surface of the cornea, usually in a surgical procedure. External ear • 1. Use swab to collect specimen from ear canal or to sample drainage. 2. Squeeze sponge to moisten swab in culturette sleeve and submit to lab.
TRANSPORT OF SPECIMENS: • A. Deliver to the lab within 2 hours of collection. Since overgrowth of the slower growing pathogenic fungi by contaminating bacteria and/or fungi is common, it is important to transport specimens to the lab as soon as possible. • B. If transport is delayed, store specimens under refrigeration at 4o C with the following exceptions: ♦ Hold blood, bone marrow and CSF at body temperature at 30-37 degree centigrade ♦ Hold dermatological specimens at room temperature. ♦ Be aware that viability may decrease with prolonged storage. C. As always, transport specimen to the lab in a biohazard bag.
References: • Clinical Microbiology Procedures Handbook, 1992, Isenberg, American Society for Microbiology. Manual of Clinical Microbiology, 1991, 5th edition, Balows, Hausler, American Society for Microbiology. Bailey & Scott's Diagnostic Microbiology, 1986, 7th edition, Finegold, Martin, Scott, C.V. Mosby Company

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Specimen collection in mycology

  • 2. COLLECTION OF SPECIMEN GENERAL PRINCIPLE The most important steps for the isolation of fungus are: • proper collection of specimens. • rapid transport to the lab. • prompt and correct processing of the specimen. • inoculation onto appropriate media. • incubation at suitable temperatures. Each specimen submitted must be examined for quality and the importance of proper collection can not be overemphasized. Fungi normally live a saprophytic existence in nature, capable of a life cycle without a human or animal host. Humans become an accidental host by inhalation of spores or by introduction of the fungus into tissue through trauma. Generally, humans are relatively resistant to these infections, but due to long- term catheterization, GI surgical procedures, chemotherapy and alterations of the immune system, exposure of fungi normally considered to be nonpathogenic can lead to infection. .
  • 3. MICROSCOPIC EXAMINATION: • A. Specimens of any clinical nature can be examined microscopically for fungi upon the physician's request or at the initiative of the laboratorian. • All specimens of sufficient quantity submitted for fungal culture should be examined microscopically for fungi. • When there is not a sufficient quantity of a specimen for both a culture and direct exam, the culture takes priority over the smear because it is more sensitive than microscopic examination. • Observing a fungus in a clinical early information that may be crucial for determining appropriate therapy for the patient.
  • 4. MICROSCOPIC EXAMINATION: Fungus stains are determined by the type of specimen. • India ink - CSF • KOH wet prep - Hair, nails, and skin scrapings • Gram stain - All other specimens, but a fungus stain performed by the histology section of the laboratory yields better results, particularly for tissue specimens.
  • 5. MICROSCOPIC EXAMINATION OF INDIA INK PREPARATION
  • 6. INDIA INK PREPARATION It is usually employed to detect the presence of microorganisms, particularly yeast, in a body fluid. It induces negative staining by creating a black, semi-opaque background that makes the capsule of the yeast clearly visible. One drop of cerebrospinal fluid (CSF) is mixed with a drop of India Ink on a clean micro slide. A cover slip is placed over it and the sample is examined through the low power lens, and then through the high power lens, of a microscope. The preparation can be diluted with a small drop of water if it is too dark and murky.
  • 7. KOH & FUNGUS CULTURE COLLECTION PROCEDURE • Hair • Scrape the scalp with a blunt scalpel. (Hair stubs, contents of plugged follicles, and skin scales are also acceptable) • Hair may also be plucked from the scalp with forceps. (Do not submit cut hair.) • Place in a dry sterile container. • Transport immediately to the laboratory at room temperature. • Nails • Cleanse the nail with alcohol. • Remove the outermost layer by scraping with a scalpel. • Clippings from any discolored or brittle parts of the nail may be used. • Deeper scrapings and debris under the edges of the nail are also suitable. • Place the specimens in a dry sterile container. • Transport immediately to the laboratory at room temperature. • Skin • Cleanse the skin with alcohol. • Collect epidermal scales with a scalpel at the active border of the lesion. • Place the specimens in a dry sterile container. • Transport immediately to the laboratory at room temperature.
  • 8. FUNGUS CULTURE COLLECTION PROCEDURE SCALP AND HAIR • Scrape the scalp with a blunt scalpel. • Collect the basal portion of the infected hair. • Note: Infected hairs may be selected by placing the patient under a UV light (Wood’s lamp). • Hairs infected with some dermatophytes will fluoresce under UV light. • Hairs that are fluorescent, distorted or fractured should be cultured. • Place hair in a dry sterile container. • Transport to the laboratory at room temperature. • The following specimens are also acceptable: 1. Hair stubs 2. Contents of plugged follicles 3. Skin scales 4. Hair plucked from the scalp with forceps
  • 9. FUNGUS CULTURE COLLECTION PROCEDURE VAGINAL SWABS • 1. Using several sterile swabs, collect discharge from the vagina and place swabs in a sterile tube that can be capped. • This method of collection provides a specimen that is also suitable for performance of a wet prep. • 2. Alternatively, Vaginal material may be collected and submitted on a culturette SKIN AND INTERSPACES • 1. Wipe lesions and interspaces between toes with an alcohol sponge. • 2. Scrape the entire lesion(s) and both sides of the interspaces with a sterile scalpel blade. • 3. Place all scrapings into a sterile urine culture cup
  • 10. FUNGUS CULTURE COLLECTION PROCEDURE TISSUE • 1. Biopsy aseptically from both the center and edge of the lesion. • 2. Place tissue in a sterile container and if transport is delayed, it may be covered with a minimum of sterile saline to prevent drying. RESPIRATORY SPECIMENS OTHER THAN SPUTUM • 1. These include bronchial washings, lung biopsies, and tracheal aspirates. • 2. These specimens are collected aseptically by the physician, placed in the appropriate container and immediately transported to the lab
  • 11. FUNGUS CULTURE COLLECTION PROCEDURE OTHER SOURCES (Submit fluid or culture swab) • 1. Bone Marrow – collect 0.5 ml aspirated material • 2. Blood – collect 2 sets of blood cultures (40 ml of blood) • 3. Corneal scraping, intraocular aspiration, or biopsy obtained by an ophthalmologist. • 4. Sputum – collect first early morning deep cough sample after patient’s teeth are brushed and the mouth is well rinsed (24 hr. specimens are not satisfactory). • 5. Subcutaneous Specimens (wound, abscess, lesion, pus, drainage) - For suppurative lesions of the deep skin and subcutaneous tissue, where pus may be loculated within abscess or is exuding from deep sinus tracts, aspiration with a sterile needle and syringe should be attempted. The material should be placed in an anaerobic transport. In addition to a fungus culture, both anaerobic and aerobic cultures should be performed. The former being necessary to recover the anaerobic branching filamentous bacteria belonging to the genus Actinomyces. • 6. Tissue – obtained in surgery. Place on sterile 4x4 gauze or in sterile container and transport to the laboratory immediately. • 7. Body Fluids and Exudates – samples are usually obtained by aspiration with a sterile needle and syringe. • 8. Urine – collect first morning “mid-stream” specimen. Do not obtain urine samples from a collection bag or bedpan. • 9. Stool – rarely worth culturing – growth of a large amount of yeast has possible significance, but only in indicating a lack of normal flora.
  • 12. TYPES OF SPECIMENS… Body fluids (pleural, synovial, peritoneal) • 1. Cleanse proposed puncture site with alcohol, followed by iodine solution. • 2. Using needle and syringe, aspirate aseptically and place in sterile container. Abscess, pus, exudate, drainage 1. Using needle and syringe, aspirate specimen and place in sterile container. 2. 2. Small volume specimens may be submitted in the syringe, without the needle. Recap the syringe with the syringe cap.
  • 13. TYPES OF SPECIMENS… Eye Swabs • 1. Notify the lab before beginning procedure. • Media will be provided by laboratory to be inoculated at the bedside. 2. For suspected mycotic keratitis, an ophthalmologist will scrape the surface of the cornea, usually in a surgical procedure. External ear • 1. Use swab to collect specimen from ear canal or to sample drainage. 2. Squeeze sponge to moisten swab in culturette sleeve and submit to lab.
  • 14. TRANSPORT OF SPECIMENS: • A. Deliver to the lab within 2 hours of collection. Since overgrowth of the slower growing pathogenic fungi by contaminating bacteria and/or fungi is common, it is important to transport specimens to the lab as soon as possible. • B. If transport is delayed, store specimens under refrigeration at 4o C with the following exceptions: ♦ Hold blood, bone marrow and CSF at body temperature at 30-37 degree centigrade ♦ Hold dermatological specimens at room temperature. ♦ Be aware that viability may decrease with prolonged storage. C. As always, transport specimen to the lab in a biohazard bag.
  • 15. References: • Clinical Microbiology Procedures Handbook, 1992, Isenberg, American Society for Microbiology. Manual of Clinical Microbiology, 1991, 5th edition, Balows, Hausler, American Society for Microbiology. Bailey & Scott's Diagnostic Microbiology, 1986, 7th edition, Finegold, Martin, Scott, C.V. Mosby Company