(2) Collection, Handling, Processing of Clinical Mycology Specimens Flashcards
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Enumerate the Specimens to revcover fungi
- Hair, skin, and nail scrapings
- Respiratory tract secretion
- Cerebrospinal fluid (csf)
- Blood
- Vaginal secretion
- Urine
- Tissue, bone marrow, and sterile body fluid
Specimen to recover fungi
Some of the most common specimens used for diagnostic mycology
Hair, skin, and nail scrapings
Specimen to recover fungi
They are commonly used to detect dermatophytes, which are keratin-loving fungi.
Hair, skin, and nail scrapings
Specimen to recover fungi
Hair samples are collected through ____, and placed on a glass slide,or if being transferred, place it initially on a paper or envelope
plucking
Specimen to recover fungi
Skin scrapings are collected with the use of a sterile blade wherein the site of collection is first cleaned with a ____ to remove normal flora, and then followed by scraping the skin.
70% alcohol
Specimen to recover fungi
When the skin has lesions, sample is collected
from?
the edge of the lesion where the most active fungi are located
Specimen to recover fungi
how nails are collected?
nail clippers
Preferably, the nails with lesions should be collected
Specimen to recover fungi
Used for detecting systemic mycoses which mainly attacks lungs
Respiratory tract secretion
Specimen to recover fungi
- It may involve sputum, bronchial washing, bronchoalveolar lavage, and tracheal aspirates.
- They are considered as non-sterile and may consist of normal flora apart from the target causative aent.
Respiratory tract secretion
Specimen to recover fungi - Respiratory
To prevent interferences from the normal flora, culture media containing inhibitors, specifically?
antibiotics
Specimen to recover fungi
- Collected by physicians through spinal or lumbar tap.
- It is considered as a sterile specimen, and no normal flora are present within the samples. (culture without inhibitors can be used)
Cerebrospinal fluid (CSF)
used
Specimen to recover fungi
what portion of CSF samples are tested?
Sediment part
Specimen to recover fungi
how sediment part of CSF isolated?
- First Method: membrane filter is attached in the syringe and is used to separate the sediment
- Second Method: centrifugation
the liquid portion should not be thrown, and should be kept at room temperature
Specimen to recover fungi
Also mainly used to detect systemic fungi
BLOOD
Specimen to recover fungi
how is blood samples examined?
automated machines
most common automated machines used in the laboratory are BACTEC, VERSATEK, and Bact-ALERT - mainly used to detect yeast
Specimen to recover fungi
- Commonly used in detecting infections mainly caused by the Candida species.
- Samples are collected through a cotton swab and are processed immediately as it can dry out which disintegrates the fungi.
ASAP Procesing
Vaginal Secretion
Specimen to recover fungi
- Processed immediately, within 24 hours, upon receipt.
- The sediment is also the portion examined and may be collected through centrifugation
Urine
Specimen to recover fungi
- Processed immediately upon receipt.
- Time of collection is noted since the optimum time of recovery of fungi is within an hour after collection
Tissue, Bone marrow, and sterile body fluids
Specimen to recover fungi
Samples should be submitted in the laboratory and
processed as soon as possible.
BUT WHAT IF THERE IS A DELAY? what can u do
Refrigeration for short time, max of 24 hours
- EXCEPT for five specimens— hair, nail, skin, blood, and CSF. Stored at RT or incubaterd at 30 ‘C
- When storing exceeds 24 hours, samples should be recollected.
quit yo job
Enumerate the methodologies for fungal infection
- Microscopic Examination (Direct and Staining)
- Fungal Culture
- Biochemical Tests
- Serology-Antigen Detection Tests
- One of the routine analysis in the laboratory
- Provides rapid diagnosis of some fungal infections. HOWEVER just the presence or absence of fungi
- Look for fungal structures
Microscopic Examination
T or F
- Direct examination require STAINS to be added
- Samples are examined in HIGH LIGHT
Both F
- Does not require stain - COLORLESS
- Examined under low light
Direct Examination
- Examination is done in a natural environment.
- Good for examining YEASTS.
- Only incorporates saline.
Saline Wet Mount
structure.
Report as either “positive” or “negative” for fungal
Direct Examination
Commonly done on skin scrapings, hair, nails, sputum, and
vaginal secretions.
Potassium Hydroxide (KOH) Preparation
Direct Examination
What is the function of KOH preparation?
softening, digesting, and clearing cellular debris, such as tissue cells and mucous, to prevent
interferences
Direct examination
what percentage of KOH are used for routine and hard exterior
- 10% KOH
- 20% KOH
Direct Examination
KOH preparation shoud be place in a petri dish together with?
Damp piece of filter paper
to prevent speciment from drying out
Report as either “positive” or “negative” for fungal
Direct Examination
Aids in the visualization of diagnostic features of fungi, especially those that are dimorphic
Stains
Enumerate all the stains
- Lactophenol Cotton Blue (LPCB)
- Calcofluor White
- India ink
- Giemsa or Wright Stain
- Gram Stain for Yeasts
- Periodic Acid Schiff (PAS)
- Gomori Methenamine Silver Stain
Stains
It is one of the most common stains used in the laboratory during routine tests
Lactophenol Cotton Blue (LPCB)
Stains
Enumeate the component of LPCB
- Lactic Acid
- Phenol
- Cotton Blue (aman stain)
Stains - LPCB
used as a preservative for LPCB
Lactic acid
Stains
Killing or removal of non-fungle element for LPCB
Phenol
Stains
the actual stain for LPCB
Cotton blue or Aman stain
Stains
- Fluorescent dye that binds with the chitin in the fungal cell wall.
- Requires a fluorescence microscope
Calcofluor White
Stains
what is the appearance of Calcfluor white stained fungi under a micrscope
appear white to blue to green fluorescence
Stains
what can be mixed with Calcofluor white for rapid diagnosis of fungal structure
KOH
stains
A negative or an indirect stain used
for the detection of the CAPSULE of
Cryptococcus neoformans in CSF
specimen
India ink
Stains
what is the causative agent of meningitis, and give the stain for the detection
- Cryptococcus neoformans
- India ink
Stains
what will be the appearance of capsule with india ink stains
Appear as clear halo against a black background
Stains
Used for the examination of bone marrow or peripheral blood sample
Giemsa or Wright stain
Stain
what does the Giemsa stain detect?
Histoplasma capsulatum—a type of systemic fungi
Stain
- Routine stain for the detection of bacteria but can also detect
fungi if present in the specimen. - Yeast stains as gram (+) which is indicated by a purple
color.
Gram Stain for Yeasts
Stain
- Stains the polysaccharide in the cell walls of fungi
- Fungi stain pink-red with blue nuclei
- Most commonly used for tissue samples collected through biopsy
Periodic Acid Schiff (PAS)
Stain
Silver nitrate outlines fungi in black due to the silver precipitating on the fungal cell wall.
Gomori Methenamine Silver Stain
Stain
what are the staining color of the internal parts of the hypae and background for Gomori stain
- Deep rose to black
- Light green
Considered to be the GOLD STANDARD for the diagnosis of fungal infection
it detects specific GENUS and SPP
Fungal Culture
TOF - Fungal Culture
Advantage of yielding the SPECIFIC ETIOLOGICAL AGENT through fungal culture—in vitro using artificial media
T
Fungal Culture Considerations
- Incubation temp (most fungal)
- Growth for most fungi
- Incubation temp for Dimorphic
- Growth for rapid growers
- Cultures maintenance
a. 37 ‘C
b. 25 ‘C to 30 ‘C.
c. 1 to 2 weeks
d. 21 to 30 days
e. high-humidity environment
- B
- D
- A
- C
- E
Culture media
what media are used for anti-fungal agent which removes fungal
contaminants
Media with and without CYCLOHEXAMIDE
Culture media
What media are used for required for samples with normal flora or
non-sterile samples
with and without an ANTIBACTERIAL AGENT
Culture media
Medium may be place in a??????
plate or tube
plated media is preffered offers a larger surface for growth
What are the types of primary culture media
- Primary or General Purpose Media
- Differential Test Medium
Contains nutrients necessary for the growth of a wide variety of fungi—making it a non-selective medium
Primary or General Purpose Media
what is the most commonly used general media
- SDA - mold culture (It can also inhibit the growth of bacteria due to its acidic pH)
- BHIA - for pathogenic fungi, specifically, yeast.
Identify what Primary Media
-
General media, recommended for
most studies. - Has an acidic pH at 5.6 that inhibits most bacteria and supports the growth of majority of fungi
Sabouraud’s
Dextrose
Agar (SDA)
Identify what Primary Media
- Primary recovery of saprobic and pathogenic fungi.
- Provides a rich medium for bacteria, yeast, and pathogenic fungi.
Brain Heart
Infusion Agar
(BHIA)
Identify what Primary Media
- Primary recovery of pathogenic fungi exclusive of dermatophytes.
- The agar provides a rich medium for yeast and pathogenic fungi.
BHIA with Antibiotics
Identify what Primary Media
- Recovery of fungi from blood
- Enhances the recovery of yeasts in blood
BHI Biphasic Blood Culture
Bottle
Identify what Primary Media
- Isolation and presumptive identification of yeast and filamentous fungi.
- contains substrates that react with
enzymes produced by different organisms that result in the production of characteristic color changes.
Chromogenic
Agar
Identify what Primary Media
- Primary recovery of dermatophytes; recommended as screening medium
- Dermatophytes produce alkaline metabolites, which raise the pH and change the medium from red to yellow
Dermatophyte Test Medium
Identify what Primary Media
- Primary recovery of pathogenic fungi exclusive of dermatophytes
- Examine plates for growth. Chloramphenicol inhibits bacterial growth.
Inhibitory Mold Agar
Identify what Primary Media
- Primary recovery of saprobic and pathogenic fungi
- Growth is enhanced by a pH alkaline reaction of fungus. Chloramphenicol and antibiotics inhibit the growth of bacteria and
nonpathogenic fungi
Potato Flake
Agar
Identify what Primary Media
- Primary recovery of dermatophytes
- Inhibits bacteria and saprophytic fungi
Mycosel
Identify what Primary Media
- Primary recovery of saprobic and pathogenic fungi
- Isolates and enhances growth of pathogenic fungi
Sabouraud’s Dextrose with Brain Heart Infusion (SABHI)
Identify what Primary Media
- Primary recovery of pathogenic fungi exclusive of dermatophytes
- Enhances the recovery of Blastomyces and Histoplasma capsulatum from contaminated
specimens
Yeast Extract Phosphate Agar
This medium used to enhance pigment development, conidia production, and mold-to-yeast phase transition of dimorphic fungi
Differential Test Medium
It is able to differentiate species as it is a selective media which allows the growth of a specific fungi, or a common group of fungi
Differential Test Medium
Identify what Differential Test Medium
Detection of ascospores in
ascosporogenous yeasts
Ascospore Agar
Identify what Differential Test Medium
Identification of Cryptococcus, Trichosporon, and Rhodotorula species
Christensen’s Urea
Agar
Identify what Differential Test Medium
Identification of Candida albicans by chlamydospore production; identification of C. albicans by microscopic morphology
Cornmeal Agar with Tween 80 and Trypan Blue
Identify what Differential Test Medium
Conversion of the dimorphic
fungus Blastomyces speciesfrom
mold to yeast form
Cottonseed Conversion Agar
Identify what Differential Test Medium
Differential identification of
Aspergillus species
Czapek’s Agar
Identify what Differential Test Medium
Identification of Cryptococcus neoformans and Cryptococcus gattii
Niger Seed Agar
(Birdseed Agar)
Identify what Differential Test Medium
Detection of nitrate reduction to confirm Cryptococcus species
Nitrate Reduction Medium
Identify what Differential Test Medium
Demonstration of pigment
production by Trichophyton rubrum;
preparation of microslide vultures and sporulation of dermatophytes
Potato Dextrose Agar
Identify what Differential Test Medium
Identification of Microsporum audouinii
Rice Medium
Identify what Differential Test Medium
Identification of Trichophyton species
Trichophyton Agars 1-7
Identify what Differential Test Medium
Detection of Cryptococcus species; differentiate Trichophyton mentagrophytes from Trichophyton rubrum; detection of Trichosporon species
Urea Agar
Identify what Differential Test Medium
Identification of yeasts by
determining fermentation
Yeast Fermentation Broth
Identify what Differential Test Medium
Identification of yeasts by
determining carbohydrate assimilation
Yeast Nitrogen-base Agar