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  • IDENTIFICATION OF FUNGI

                                                                        بسم الله الرحمن الرحيم
                                                                      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. 
    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
    A.Molds (filamentous fungi)
    B.Yeasts (bacteria-like fungi) 
                   Types of fungi
    Molds (filamentous fungi)

    consist of long, branching filaments of cells called hyphae (singular hypha). 

    a mass of hyphae visible to the unaided eye is a mycelium (plural, mycelia).

     In some molds, cross walls may occur along the hypha. Those fungi that have cross walls are called septate fungi, since the cross walls are called septa. 
    Reproduction in fungi is by spores.

    Spores are produced by either sexual or asexual means. 

    Asexual spores may be free and unprotected at the tips of hyphae, where they are called conidia.

    Asexual spores may also be formed within a sac, called sporangiospores. 
    B.Yeasts 

    are microscopic.

    unicellular fungi with a single nucleus and eukaryotic organelles.

    They reproduce asexually by a process of budding 
                                                          Molds (filamentous fungi)
        Molds (filamentous fungi


    Aspergillus




    Aspergillus






    Rhizopus


    Rhizopus






    Penicillium






    Penicillium
    Penicillium






    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 is used to culture of certain types of fungi 
    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 day





    Other Diagnostic Tools
    API Commercial System




    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
    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 Accessories


        


    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. 
    PIC
    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. 
    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.
    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.
    CYCLES
    In presence of polymerase enzyme (heat resistant) separation of a double stranded DNA (parentral strands) by heat at 95 degrees.
    o       Annealing: Synthetic pair of oligonucleotide probes are allowed to attach to their matching base sequences on the separated DNA helicals.
    o       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-.
    o       This 3 step cycle is repeated 25 to 35 times.




















2 التعليقات:

  1. Anonymous says:

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  1. Anonymous says:

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