Yeast - Major Characteristics Unicellular Fungi Eukaryotic Facultative anaerobes Capable of formingcolonies on solid culturemedia. Occur worldwide Over 1,500 speciesdescribed
They reproduce either asexually (most common) or sexually.•Asexual reproduction is through budding or binary fission.•Sexual reproduction (if any) results in the formation of theappropriate spore structure.BuddingYeast - ReproductionFissionSporesSaccharomyces cerevisiaeSchizosaccharomyces octosporus
Yeast Significance Food Industry• Fermentation of bread, and alcoholic beverages.E.g. Saccharomyces cerevisiae (also called baker’s yeast or sugaryeast) used in baking. Medical• E.g. Candida albicans - common in the human mouth, but canbecome pathogenic and cause Candidiasis (oral and/or genitalinfection). Biofuel Industry•Production of ethanol for car fuel.
Principal Criteria and Tests forIdentifying Yeasts1. Culture characteristics - Colony color, shape,texture2. Asexual structuresa. Shape and size of cellsb. Fission, bipolar, multipolar or unipolarbudding.c. Absence or presence of germ tubes, hyphae,or pseudohyphae.
The ascocarp in this class is a- Spherical.- Complete closed structure calledcleistothecium.- It has no opening to release asci. The classincluded two orders:• Include two Orders:•Eurotiales (Saprophytic Plectomycetes).•Erysiphales (Parasitic Plectomycetes).
Order: Eurotiales:Saprophytic plectomycetsAsci globos and scattered withincleistotheciumAspergillus spp. are examples of thisorder
Order: Erysiphales:* Parasitic plectomycetes on vascular plant.* Cause a disease called powdery mildew.* Cleistothecia formed on superficialmycelium and asci are cylindrical.
Identification the genera of Erysiphales(Powdery Mildew Fungi)- Powdery mildews are caused by several genera ofAscomycotina- They are host specific, biotrophic, andobligately parasitic.- Fungus produces hyphae and conidia usually atthe upper surface, or on stems, flowers, orfruit.- They can be identified most easily by the formationof ascocarp with no natural opening, called acleistothecium.
MATERIALSFresh and dried plant materials with powdery mildewsignsDissecting needles/ single-edge razor bladesMicroscope slidesCoverslipsPROCEDURES- Asexual stage- Sexual stage
Key to Genera of Powdery Mildew FungiAppendages coiled or hooked at tip – UncinulaAppendages simple and straight with bulb-like base– PhyllactiniaAppendages branching dichotomously at tipCleistothecium contains a single ascus –PodosphaeraCleistothecium contains several asci –MicrosphaeraAppendages simple or irregularly branched, oftenentwinedCleistothecium contains a single ascus – SphaerothecaCleistothecium contains several asci – Erysiphe
These species have septate, mycelium.Produce asci and ascospores that are borne inan ascocarp.There are four types of ascocarps recognized;cleistothecium, perithecium, apotheciumand ascostroma.Species that produce cleistothecia, peritheciaand apothecia have unitunicate asciwhile those that produce ascostroma havebitunicate asci.
A unitunicate-operculate ascus has a:"lid", the Operculum, whichbreaks open when the sporesripen and in this way setsthem free.Unitunicate-operculateasci only occur in those ascocarpswhich have apothecia, Unitunicate meanssingle-walled.
Mechanism of ascospore discharge from Unitunicate Ascus
A bitunicate ascus is enclosed in adouble wall.When the spores are ripethe shell splits open, sothat the spores can escapeinto free air. Bitunicate asciExamples: Venturia inaequalis(apple scab) and Guignardia sp.(Brown Leaf Mold of Horse Chestnut).
Mechanism of ascospore discharge from Bitunicate Ascus
Prototunicate asciare mostly spherical in shape and they haveno active dispersalmechanism at all.Asci of this type canbe found both inperithecia and incleistothecia.
Class: Plectomycetes (Cleistothecia) Class: Pyrenomycetes (perithecium) Formation of an ascocarp called a perithecium. This ascocarp is typically flask-shaped orglobose Have a small ostiole through which theascospores are released. Asci are unitunicate and are arranged, in asingle fertile layer throughout the base of theperithecium (hymenium). Ascospores are often forcibly ejected from theascus and perithecium when mature.
Order: SordarialesThe perithecia are usually dark orpallid, flask-shaped ascocarps with anostioleparaphyses are absent when ascosporesare mature.Sordaria fimicola: This is an example of aspecies that does not produce a stroma.In nature, this species grows on dung.
Order: Xylariales This order includes a very large and diverse groupof Pyrenomycetes that typically produce theirperithecia in stromata. Xylaria sp. and Penzigia sp. Their stromata areexternally black, and can be seen to be mostlywhite in section. The perithecia are entirely immersed in thestromata with only the ostioles opened to thesurface. The stromata of Xylaria are long and taperingwhile those of Penzigia sp. are hemisphaericalto globose that usually occur in clusters.
Stromata of XylariaStromata of Penzigia globosum
Class: Discomycetes The series Discomycetes is characterized by the formationof an ascocarp called an apothecium. Typically an apothecium is cup-shaped which is whyDiscomycetes are sometimes called "cup fungi". The asci are unitunicate and forcibly eject theascospores. Paraphyses are generally present in apothecia. Morchella sp. a species in which the apothecium hasnow formed depressions that are fertile with sterile ridgesin between; Leotia sp. a species in which the "cup" of the apotheciumis interpreted as being folded back, Gyromitra sp. a species in which the apothecium is saidto be "saddle-shaped
General characteristics of Zygomycotina•The name comes from zygosporangia.•They are mostly terrestrial in habitat living in soil or ondecaying plant or animal material.•Zygomycete hyphae are coenocytic, forming septa onlywhere gametes are formed•Some are parasites of plants, insects, small animals andother fungi, while others form symbiotic relationshipswith plants.
Sexual and A Sexual Reproduction• Gametangial copulation• Conjugation by two morphologically similiargametangia• Production of zygospores (thick-walled restingspores) within zygosporangia that are formedby fusion of gametangia• Produce a zygosporangium• Homo- & heterothallic species
Meeting of two different mating typeZygospores
How to differentiate between Mucor and RhizopusMacroscopic Features of Mucor- Colonies of Mucor grow rapidly at 25-30°C and quicklycover the surface of the agar.- Its cottony appearance.- From the front, the color is white initiallyand becomes grayish brown in time.Macroscopic Features Rhizopus- Colonies of Rhizopus grow very rapidly at 40-45°Cfill the Petri dish, and mature in 4 days.- The texture is typically cotton-candy like.- From the front, the color of the colony iswhite initially and turns grey toyellowish brown in time.
Microscopic Features of Mucor- Nonseptate, with broad hyphae.- Sporangiophores, sporangia, and sporangiospores arevisualized.- Apophysis, rhizoid and stolon are absent.- Sporangiophores are short, erect and mayform short sympodial branches.- Columella are hyaline or dematiaceous- Sporangia are round, 50-300 µm in diameter,gray to black in color, and are filled with sporangiospores.- The sporangiospores are round (4-8 µm in diameter)or slightly elongated.
Microscopic Features Rhizopus- Nonseptate broad hyphae- Sporangiophores, rhizoids, sporangia, andsporangiospores are visualized.- Sporangiophores are brown in color and unbranchedand they can be solitary or form clusters.- Apophysis, rhizoids stolons is present.- Rhizoids are located at the point wherethe stolons and sporangiophores meet.- Sporangia (40-350 µm in diameter).- Sporangiospores (4-11 µm in diameter)are unicellular, round to ovoid in shape,hyaline to brown in color.
Genus BestgrowthSporangi-ophoreApophysis Columella Sporan-giumRhizoidMucor <37°CBranched orunbranched,hyaline-+, in varyingshapesSpherical -Rhizopus 45°CUnbranchedand brownmostlyNotprominentSpherical orelongatedSpherical +
Rhinocerebral ZygomycosisRhinocerebral mucormycosis: is a rare opportunisticinfection of the sinuses, nasal passages, oral cavity, andbrain which commonly caused by Rhizopus oryzae. Theinfection can rapidly result in death.
Homework What is the function of columella? What is Azygospore?
General characteristics There is no sexual reproduction in life cycle or thereproduction occurs by nonsexual stages. Nonsexual reproduction may occur by cellulardivision or by germination of mycelialfragments or spores of various types. The spores may be chlamydospores or conidia. Chlamydospores are formed by transformationof an intercalary somatic cell or group of cells byrounding up of the cells and deposition of a thickwall.
Classes of Deuteromycotina: Blastomycetes. Hyphomycetes. Coelomycetes.
Blastomycetes: Members of this class include yeast likefungi. which fail to produce ascospores. Others appear to have affinities withBasidiomycotina. These yeasts may be isolated from a varietyof habitate, including water, the soil, orplants. Many are pathogenic on animals or human,such as Candida.
Hyphomycetes: Include those deuteromycetes that form amycelium but lack a sporocarp (Conidiomata). In some species the conidiophores may be tightlyclustered together to form a pulvinate mass, theSporodochium or Synnema. Development of conidia from phialid: a basipetalsuccession of conidia is formed from a fixed locuson the conidiogenous cell (phialide)
Conidiogenous Cells A hyphal compartment or cell from which, or onwhich, a conidium is formed Conidiogenous cell may be formed directly from ahypha, or on a specialized simple or branchedhypha called a conidiophore.
Sporodochium of Fusarium sp.Sporodochium: A compact, cushion-like aggregation ofhyphae on which conidiophores are formed in a dense layer
Synnema in Arthrobotryum spSynnema:•Conidiophores united at base•Conidiogenous cells at apex
Coelomycetes: In this class the conidiophores are borne on orwithin a multicellular sporocarp, calledConidiomata, which has two types, Pycnidiumand Acervulus. Pycnidium is a closed sporocarp (Conidiomata)bearing its conidiophores, conidiogenous cellsand conidia within a cavity. The pycnidium may be discoid, globose or flasklike. Pycnidia may be entirely closed or may opento the outside by an ostiole.
Acervulus is an open mass of closely packedconidiophores and conidiogenous cells thatmay form a flat discoid cushion of conidia.Acervuli are usually formed on a plant hostand are often erumpent from the epidermisof the host, pushing aside flaps of hosttissue as they emerge.
Home work What is Ontogeny? What are the differences between Perithecium andPycnidium?
The type and quality of specimenssubmitted to the mycology laboratory arean initial factor in determining the successof isolating and identification of fungi.
Important steps for successful isolation of etiologicalagents of mycoses are:1. Proper collection of the specimens.2. Rapid transport of the specimens to the laboratory.3. The correct processing of the specimens.4. Inoculation of specimens onto appropriate culture mediaand incubation at suitable temperatures.
COLLECTION AND TRANSPORT OF SPECIMENS:A- Specimens should be collected aseptically, placed insterile humidified, leak-proof container, delivered tothe laboratory within 2 hours, processed, and theninoculated to primary isolation media within a fewhours of collection..
COLLECTION AND TRANSPORT OF SPECIMENS:Dermatologic specimens, however, should betransported in a dry container. Transport mediumshould not be used unless the specimen can be easilyand completely retrieved from the medium.
B - The effect of refrigeration on fungal specimens has notbeen well-studied.- If processing is to be delayed for more than severalhours, it is recommended that specimens be stored underrefrigeration at 4 ̊C.- Exceptions: blood and cerebrospinal fluid are stored at30- 37 ̊C.C- Swabs are not encouraged; however, specimens fromthe environment or certain body sites such as the earcanal, nasopharynx, and throat are not readily collectedby other means.
2. SPECIMENS:A. Sputum (tracheal lavage, and bronchial lavage)1. Sputum should be fresh and collected in the early morning??2. Sputum should be the result of a deep cough (not saliva).3. Collect 5-10 ml in sterile container.B. Respiratory specimens other than sputum, such as trachealaspirates, and lung biopsy materialC. Blood1. Blood is collected aseptically to avoid microbial contamination.2. Use sodium polyanethol sulfonate (SPS, Liquoid) as ananticoagulant.
D. Pus, Exudates - Using a sterile needle and syringe.E. Tissue1. Tissue is aseptically collected from the center and edgeof the lesion.2. Place between moist gauze squares, add a small amountof sterile water to keep tissue from drying out.F. Bone Marrow - Aspirate approximately 3-5 ml of bonemarrow and place it in a sterile container. SPS or heparincan be added as an anticoagulant.
G. Cerebrospinal Fluid - spinal fluid as possible iscollected and placed in a sterile container.H. Urine1. The urine specimen most suitable for making adiagnosis of mycoses of the urinary tract.2. Early morning specimens are aseptically collected insterile containers. Twenty-four hour collections have novalue. Urine may be stored at 4 ̊C for up to 12-14 hours
I. Body Fluids (pleural, synovial, and peritoneal).J. Hair1. Select infected areas and with forceps, and take at least 10 hairs.2. Place hairs between two clean glass slides or in a clean envelopelabeled.K. Nail1. Clean nail with 70% alcohol.2. Scrape outer surface and discard.3. Collect whole nail or nail clippings.4. Place all material in a clean envelope labeled with the patientsdata.
L. Skin and Interspaces1. Clean the lesions and interspaces between the toeswith alcohol sponge or sterile water.2. Scrape the entire lesion(s) and both sides ofinterspaces with a sterile scalpel.3. Place scrapings between two clean glass slides orplace in a clean envelope labeled with the patients data.
EXAMINATION OF SPECIMENSMACROSCOPIC AND MICROSCOPIC EXAMINATION:1. MACROSCOPIC EXAMINATION:Before inoculating a specimen to the appropriateisolation media, the specimen is examinedmacroscopically for bloody areas, and necroticmaterial.Specimens from cases of mycetoma areexamined with the dissecting microscope for thepresence of granules before proceeding
1.Potassium Hydroxide Procedure (KOH Examination)KOH may be used to examine hair, nails, skin scrapings, fluids, orbiopsies. The fungal structures such as hyphae, large yeast(Blastomyces), and sporangia may be distinguished.2. MICROSCOPIC EXAMINATION:
1. Potassium Hydroxide Procedure (KOH Examination)…continued Specimens placed in a drop of 15% KOH willdissolve at a greater rate than fungi because fungi havechitinous cell walls. Fluids such as CSF generally do not need to betreated with KOH. The clearing effect can be accelerated by gentlyheating the KOH preparation. Visualization of fungi can be further enhanced by theaddition of Parker Superquink permanent black inkto the preparation.
2- India Ink Examination:India ink can be added to specimens such asspinal fluids or exudates to provide a darkbackground that will highlight hyaline yeast cellsand capsular material.
Objectives What is Candida albicans ? Applying Germ tube test, Chlamydosporeproduction, Temperature tolerance techniques todifferentiate Candida albicans from other Candidaspecies. Using API 20 Candida and CHROMagar Candidato differentiate between Candida species.
Aim To understand the clinical significance of Candida albicans. To explain some practical approaches for identification ofCandida albicans. Also applying some techniques to differentiate betweenCandida species.
What is Candida albicans?• Candida albicans is a yeast growth present in all of usand is normally controlled by bacteria in the intestines.• But when something destroys helpful bacteria, the yeastbegins to invade and colonize the body tissues.
What is Candida albicans? continued..• These yeast colonies release powerful chemicals into thebloodstream, causing such varying symptoms as lethargy,chronic diarrhea, bladder infections, muscle and joint pain,and severe depression.• The medical term for this yeast overgrowth is candidiasis
Isolation of Candida albicans:•Insert the cotton end of each swab into 0.5 ml ofsterile water in a micro centrifuge tube.•Mix the tube for 30s with a laboratory tabletopvortex mixer.•Spread 0.15 ml of the wash onto plates containingSabouraud dextrose agar or (PDA).•Incubate plates at 37C for 48 h..
Identification of Candida albicans:1- Germ Tube Test: This is a rapid test for thepresumptive identification of C. albicans- Reagents / Materials / Media•Bovine or human serum - A small volume to be used as aworking solution may be stored at 2 to 8 0C .•Stock solution can be dispensed into small tubes and stored at -20 0C .•Clean glass microscope slides•Glass cover slips•Glass tubes•Pasteur pipettes
- ProcedurePut 3 drops of serum into a small glass tube.Using a Pasteur pipette, touch a colony of yeast and gentlyemulsify it in the serum. The pipette can be left in the tube.Incubate at 350C to 370C for up to 3 hours but no longer.Transfer a drop of the serum to a slide for examination.Coverslip and examine microscopically using x 40 objective.Positive test: presence of short lateral filaments (germ tubes) onepiece structureNegative test: yeast cells only (or with pseudohyphae) always twopieces
2- Analytical profile index (API 20 Candida):• API is a classification of bacteria and fungi based onexperiments, allowing fast identification.• The API 20E/C fast identification system combines someconventional tests and allows the identification of bacteria andfungi.• The test systems are stored in limited small reaction tubes, whichinclude the substrates.
CHROMagar Candida appears to be a medium well-suitedfor medical mycological use. It is a new differential culture medium that is claimed toease the isolation and presumptive identification ofsome clinically important yeast species. It is also use as an adjunctive differential medium for theidentification of yeasts isolated on other media.3- CHROMagar
CHROMagar Candida components: peptone (10 g/liter), glucose (20 g/liter), agar (15 g/liter), the medium contained chloramphenicol (0.5 g/liter) chromogenic mix (2 g/liter). The medium was prepared according to themanufacturer’s instructions and dispensed into petridishes (20 ml into 100-mm-diameter dishes).
4- Chlamydospore production Corn Meal Agar (CMA) is a culture medium used aspresumptive test for identification of Candida albicans. Chlamydospore production on CMA is a characteristic featureof Candida albicans.
Procedure 17 g of Corn Meal Agar incorporated with Tween 80 isprepared according to manufacture’s instruction. Yeast colonies would then be inoculated on the CMA. Plates will then incubate at 25 ̊C for 72 hrs. Chlamydospore production would be examined after stainingwith lactophenol cotton blue under microscope.
5- Temperature tolerance Several Candida species can be differentiated by the abilityto grow at different temperatures. C. albicans can grow at 37 °C and 45 °C, while C.dubliniensis is only able to grow at 37 °C.
Objectives What is mycotoxin? What are the types, toxicity and mode of actionmycotoxins? How to remove mycotoxin?
Aims To provide a basic background about mycotoxins, theirtypes, toxicities, and mode of actions. To explain how to prevent cereal from contamination bymycotoxins.
What is mycotoxin? The term mycotoxin is a combination of Greekword “mykes” means fungus and Latin word“toxicum” meaning by poison. Mycotoxins are secondary metabolites ofmoulds that exert toxic effects on animals andhumans. The toxic effect of mycotoxins on animal andhuman health is referred to as mycotoxicosis.
Some mycotoxins were heat stable up to as much as400°C. Consumption of a mycotoxin contaminated diet may resultin teratogenic, carcinogenic, and/or immune-suppressiveeffects.
Outbreak Outbreaks occur in groups because of a sharedcontaminated food supply and the optimal weatherconditions for Aspergillus growth First recorded outbreak was in England in 1962, where100000 turkeys died.
Common mycotoxins Zearalenone Aflatoxin Fumonisins Ochratoxin Trichothecenes Nivalenol
ZearalenoneSource: F. graminearum, F. culmorum, F. equiseti,and F. crookwellense.Toxicity: It causes of a reproductive disorder in pigs known as vulvo-vaginitis. It has carcinogenic effect. The mode of action: it is an estrogenic mycotoxin which is involved inreproductive disorders and hyperestrogenicity in farm animal because ofits structural similarity with estradiol.
AflatoxinSource: Aspergillus flavus A. parasiticus, A. bombycis, A.ochraceus, A. nomius and A. Pseudotamari.Toxicity: pulmonary carcinogen, acute aflatoxicosis.Mode of Action:Cytochrome P450 enzymes convert aflatoxins to thereactive 8, 9-epoxide form which is capable ofbinding both DNA (inducing point mutation and DNAstrand break) and proteins.
FumonisinsSource: Fusarium verticillioides (formly F. moniliforme),F. proliferatum, F. nygamai and Alternaria.Toxicity: toxicosis in swine, equine leukoencephalopathyMode of Action: disruption of sphingolipid because of structural similaritywith sphingoid bases (sphinganine and sphingosine) Free sphingoid bases are toxic to the most cells byaffecting cell proliferation and inducing apoptosis ornecrotic cell death.
OchratoxinSource: A. ochraceus, A. alliaceus, A. auricomus, A.carbonarius, A. glaucus, A. melleus and A. niger.Toxicity: nephrotoxic, hepatotoxic, teratogenic inlaboratory animals.Mode of Action: inhibiting the phenylalanine-tRNAligase complex which involves in the synthesis ofphenylalanine and inhibits mitochondrial ATPproduction.
TrichothecenesSource: Fusarium, Trichoderma, Trichothecium,Myrothecium, Stachybotrys.Toxicity: weight loss, vomiting, bloody diarrhea,severe dermatitis, hemorrhage, decreased eggproduction, abortion, and death in animals.Mode of Action: it is a potent inhibitors of protein,DNA, RNA synthesises and interact with cellmembrane.
NivalenolSource: F. cerealis and F. poae , F. culmorum and F. graminearum.Toxicity: embryo toxic and fetotoxic.The mode of action: inhibitor of protein, RNA, DNAsynthesis in mammalian cells, necrosis of theproliferating cells in vivo,
Food contamination by mycotoxinCereal plants may be contaminated by mycotoxins in two ways: Fungi growing as pathogens on plants. Or fungi may grow saprophytically on stored plants.
How to control mycotoxicosis?Three methods can be used to remove mycotoxin fromcontaminated diets:1. Physical method (using adsorbents such as activated carbon andbentonite).2. Chemical method (calcium hydroxide, ozone or ammonia).3. Biological method (conversion of aflatoxin B1 (particularly byFlaobacterium auranticum) to harmless degradation products).