IDENTIFICATION OF FUNGI
تحديد نوع الفطريات
IDENTIFICATION OF FUNGI
Specimen collection
Specimens for fungal microscopy and culture may be:
• Scrapings of scale, after cleaning with alcohol.
• Skin stripped off with adhesive tape, which is then stuck on a glass slide.
• Hair which has been pulled out from the roots.
• Brushings from an area of scaly scalp.
Specimen collection
Specimens for fungal microscopy and culture may be:
• Nail clippings.
• Skin biopsy.
• Moist swab from a mucosal surface (inside the mouth or vagina) in a special transport medium.
• A swab should be taken from pustules in case of secondary bacterial infection.
Morphology
Direct Microscopy
• Potassium hydroxide (KOH) stained with blue or black ink.
• Unstained wet-mount.
• Stained dried smear.
• Histopathology of biopsy with special stains.
Types of fungi
Molds (filamentous fungi)
Molds (filamentous fungi)
Aspergillus
Rhizopus
Penicillium
on lemon
Candida albicans (yeast)
• The ability of Candida albicans to convert from the yeast (Y) form to mycelial forms through germ tube (GT) formation is considered a key feature of the transition of the organism from commensalism to virulence.
Candida albicans
Candida albicans
Culture
• Sabouraud's Dextrose agar:
• Used to culture fungi
• High %age of glucose
• Has a low pH that inhibits the growth of most bacteria;
• May also contains the antibiotic gentamicin to specifically inhibit the growth of Gram's-negative bacteria.
• Potato Dextrose agar; (PDA)
• PDA is used to culture of certain types of fungi
Culture
Cultivation of fungi is mostly at lower temperature and longer time than that for bacteria.
Temperature preferably at room temperature (~24oC).
Time for incubation 3 – 14 days.
Other Diagnostic Tools
API Commercial System
A set of tubes
containing
media for biochemical reactions
API Commercial System
• API is an abbreviation for a company called Analytab Products Inc.
• They produce a variety of commercial kits are availabe for the diagnosis of bacteria of medical significance, mainly in the family Enterobacteriaceae.
• Most of these kits consist of 6 to 21 tests which give a pattern of results allowing bacterial identification to the genus or species level.
API Commercial System
• With API, bacterial identification became simple, rapid and reliable.
• Today, API/ID32 is the most extensive range available.
• It includes 15 identification systems covering all groups of bacteria and over 600 different species encountered microbiology laboratories.
API Commercial System
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction
Definition
• Is a technique widely used in molecular biology.
• Used for quickly "cloning" a particular piece of DNA in the test tube, and
• searching for genes of interest
• It can only replicate fairly small pieces of DNA
Polymerase Chain Reaction Application of PCR
• PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas.
• PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria or viruses.
• The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
• PCRs have a variety of uses, including “genetic fingerprinting” at the crime scene.
• This is often critical for forensic analysis,, when only a trace amount of DNA is available as evidence.
PCR machine (thermal cycler)
PCR machine
PCR Accessories
Polymerase Chain Reaction (PCR)
DNA amplification takes place in small tubes placed into a PCR machine.
It is basically a heating and cooling block.
The mix in the tubes initially contains a small amount of Genomic DNA, Taq polymersae, primers, salts, buffers, and extra G's, A's, T's and C's that are added on while the DNA pieces are built.
Once the cycles finish we visualize the reactions to see if they worked by running a small amount out on an agarose gel.
Polymerase Chain Reaction (PCR)
The mix in the tubes initially contains a small amount of
• Genomic DNA, DNA segment to be copied
• Taq polymersae, extracted from a deep-sea, thermal bacterium, Thermus aquaticus.
• primers,
• salts,
• Buffers,
• DNA nucleotides to serve as feedstock .
Polymerase Chain Reaction (PCR) (for reading)
• The reaction mixture for a PCR test contains the target DNA to be amplified, two primers, and heat-stable Taq polymerase.
• Nucleic acid from the m.o. (double-stranded DNA) is denatured by heating to 95°C.
• Two short single-stranded primers (DNA fragments that are complementary) bind to the separated DNA strands when the temperature is lowered to 55°C. The primers are extended with Taq polymerase which brings in new nucleotides to build new DNA strands at 72°C.
Polymerase Chain Reaction (PCR)
(for reading)
• The first synthesis cycle results in two copies of the target DNA sequence. The cycle is repeated (denaturation, annealing, and extension), and the second synthesis cycle results in four copies of the target DNA sequence. The billions of copies of the new DNA strands that are produced during 40 PCR cycles are detected using gel electrophoresis.
Polymerase Chain Reaction (PCR)
• In presence of polymerase enzyme (heat resistant) separation of a double stranded DNA (parentral strands) by heat at 95 degrees.
• Annealing: Synthetic pair of oligonucleotide probes are allowed to attach to their matching base sequences on the separated DNA helicals.
• Since DNA polymerase is not denaturated by heat, its presence will allow the small synthesized oligonucleotide probe to extend along the specific DNA fragment – sort of replication-.
• This 3 step cycle is repeated 25 to 35 times.
Specimen collection
Specimens for fungal microscopy and culture may be:
• Scrapings of scale, after cleaning with alcohol.
• Skin stripped off with adhesive tape, which is then stuck on a glass slide.
• Hair which has been pulled out from the roots.
• Brushings from an area of scaly scalp.
Specimen collection
Specimens for fungal microscopy and culture may be:
• Nail clippings.
• Skin biopsy.
• Moist swab from a mucosal surface (inside the mouth or vagina) in a special transport medium.
• A swab should be taken from pustules in case of secondary bacterial infection.
Morphology
Direct Microscopy
• Potassium hydroxide (KOH) stained with blue or black ink.
• Unstained wet-mount.
• Stained dried smear.
• Histopathology of biopsy with special stains.
Types of fungi
Molds (filamentous fungi)
Molds (filamentous fungi)
Aspergillus
Rhizopus
Penicillium
on lemon
Candida albicans (yeast)
• The ability of Candida albicans to convert from the yeast (Y) form to mycelial forms through germ tube (GT) formation is considered a key feature of the transition of the organism from commensalism to virulence.
Candida albicans
Candida albicans
Culture
• Sabouraud's Dextrose agar:
• Used to culture fungi
• High %age of glucose
• Has a low pH that inhibits the growth of most bacteria;
• May also contains the antibiotic gentamicin to specifically inhibit the growth of Gram's-negative bacteria.
• Potato Dextrose agar; (PDA)
• PDA is used to culture of certain types of fungi
Culture
Cultivation of fungi is mostly at lower temperature and longer time than that for bacteria.
Temperature preferably at room temperature (~24oC).
Time for incubation 3 – 14 days.
Other Diagnostic Tools
API Commercial System
A set of tubes
containing
media for biochemical reactions
API Commercial System
• API is an abbreviation for a company called Analytab Products Inc.
• They produce a variety of commercial kits are availabe for the diagnosis of bacteria of medical significance, mainly in the family Enterobacteriaceae.
• Most of these kits consist of 6 to 21 tests which give a pattern of results allowing bacterial identification to the genus or species level.
API Commercial System
• With API, bacterial identification became simple, rapid and reliable.
• Today, API/ID32 is the most extensive range available.
• It includes 15 identification systems covering all groups of bacteria and over 600 different species encountered microbiology laboratories.
API Commercial System
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction
Definition
• Is a technique widely used in molecular biology.
• Used for quickly "cloning" a particular piece of DNA in the test tube, and
• searching for genes of interest
• It can only replicate fairly small pieces of DNA
Polymerase Chain Reaction Application of PCR
• PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas.
• PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria or viruses.
• The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
• PCRs have a variety of uses, including “genetic fingerprinting” at the crime scene.
• This is often critical for forensic analysis,, when only a trace amount of DNA is available as evidence.
PCR machine (thermal cycler)
PCR machine
PCR Accessories
Polymerase Chain Reaction (PCR)
DNA amplification takes place in small tubes placed into a PCR machine.
It is basically a heating and cooling block.
The mix in the tubes initially contains a small amount of Genomic DNA, Taq polymersae, primers, salts, buffers, and extra G's, A's, T's and C's that are added on while the DNA pieces are built.
Once the cycles finish we visualize the reactions to see if they worked by running a small amount out on an agarose gel.
Polymerase Chain Reaction (PCR)
The mix in the tubes initially contains a small amount of
• Genomic DNA, DNA segment to be copied
• Taq polymersae, extracted from a deep-sea, thermal bacterium, Thermus aquaticus.
• primers,
• salts,
• Buffers,
• DNA nucleotides to serve as feedstock .
Polymerase Chain Reaction (PCR) (for reading)
• The reaction mixture for a PCR test contains the target DNA to be amplified, two primers, and heat-stable Taq polymerase.
• Nucleic acid from the m.o. (double-stranded DNA) is denatured by heating to 95°C.
• Two short single-stranded primers (DNA fragments that are complementary) bind to the separated DNA strands when the temperature is lowered to 55°C. The primers are extended with Taq polymerase which brings in new nucleotides to build new DNA strands at 72°C.
Polymerase Chain Reaction (PCR)
(for reading)
• The first synthesis cycle results in two copies of the target DNA sequence. The cycle is repeated (denaturation, annealing, and extension), and the second synthesis cycle results in four copies of the target DNA sequence. The billions of copies of the new DNA strands that are produced during 40 PCR cycles are detected using gel electrophoresis.
Polymerase Chain Reaction (PCR)
• In presence of polymerase enzyme (heat resistant) separation of a double stranded DNA (parentral strands) by heat at 95 degrees.
• Annealing: Synthetic pair of oligonucleotide probes are allowed to attach to their matching base sequences on the separated DNA helicals.
• Since DNA polymerase is not denaturated by heat, its presence will allow the small synthesized oligonucleotide probe to extend along the specific DNA fragment – sort of replication-.
• This 3 step cycle is repeated 25 to 35 times.