MYCV 311 Prelim Lab - Microbiology Lab Manual PDF

MYCOLOGY AND VIROLOGY (LABORATORY) Quitaleg, Jenny Rose Maghuyop LAB PRELIM 1 CULTURE MEDIA PREPARATION AND CULTURE OF YEASTS AND MOLDS OUTLINE...

MYCOLOGY AND VIROLOGY (LABORATORY) Quitaleg, Jenny Rose Maghuyop LAB PRELIM 1 CULTURE MEDIA PREPARATION AND CULTURE OF YEASTS AND MOLDS OUTLINE I. CULTURE MEDIA I. Introduction to Culture Media II. Comparison: Agar vs. Broth Culture Media  liquid (broth) or gel ( solid/ agar) designed to support the III. Culture Medias Used In Mycology growth of microorganisms Laboratory  in Mycology are designed for detection, growth, and IV. Preparation of Agar and Broth Culture identification of yeasts and molds in clinical specimens Media  in clinical laboratory, different types of enrichment and V. Culturing Yeasts from Fruit Peels selective media is available to isolate pathogenic fungi VI. Culturing Molds from Contaminated Bread or Rice II. AGAR VS. BROTH CULTURE MEDIA Agar Media Broth Culture Media agar / x appearance at solid liquid room gelatin-like temperature appearance and texture storage petri dish bottles (screw cap) tube growth of on the surface throughout the microorganism liquid AGAR COMPONENT  causes the culture media to solidify and gives its gelatin- like appearance and texture at room temperature Page 1 of 3  Note: III. CULTURE MEDIAS USED IN MYCOLOGY o Time only starts when pressure and LABORATORY temperature are already on the desired number o Wait for the pressure and temperature to lower before opening the autoclave to  Birdseed agar avoid explosion  Brain-heart infusion agar Dispense: Cool to 45 to 50°C. Mix well before  Canavanine-glycine-bromthymol blue agar dispensing into petri dish as desired  Chromogenic agar for Candida Note:  Cornmeal agar o Always practice aseptic technique using alcohol lamp to disinfect the air and  Littman oxgall agar working area  Mycosel / mycobiotic agar Sabouraud Dextrose Agar (SDA)  Potato dextrose agar / broth Weigh: 65 grams of agar per 1000mL  Rice grains medium purified/distilled water  Sabouraud dextrose agar / broth Note: o 32.5 grams of agar per 500mL purified/distilled water Dissolve: Heat to boiling to dissolve the medium IV. CULTURE MEDIA PREPARATION completely Sterilize: Sterilize by autoclaving at 15 lbs. pressure for 15 minutes Dispense: Cool to 45 to 50°C. Mix well before dispensing into petri dish as desired MATERIALS Potato Dextrose Broth (PDB) Weigh: 24 grams of powder per 1000mL purified/distilled water  Culture Media (Granulated Agar/Broth) Dissolve: Heat to boiling to dissolve the medium completely  Distilled Water Sterilize: Sterilize by autoclaving at 15 lbs. pressure for  Erlenmeyer Flasks 15 minutes  Stirring Rod Dispense: Cool to 45 to 50°C Mix well before dispensing  Analytical Balance into tubes as desired  Cotton NOTE:  Alcohol Lamp o In some cases, when pH 3.5 is required, acidify the  Autoclave medium with sterile 10% tartaric acid (approx. 1 mL  Petri Dishes / Sterile Tubes of acid per 100 mL of media) 10 mL of tartaric acid in 1000 mL o Do not heat after the addition of acid o Acidifying the culture media will help to prevent contamination from bacteria and other contaminants V. YEAST CULTURE MATERIALS  Fruit PROCEDURE o (natural habitat of yeast because of nutrient content, orange – most recommended)  Culture Media (PDA or SDA) Potato Dextrose Agar (PDA)  Cutter Weigh: 39 grams of agar per 1000mL  Ruler purified/distilled water  Marker Dissolve: Heat to boiling to dissolve the medium  Plastic wrap completely  Shoe box Sterilize: Sterilize by autoclaving at 15 lbs. pressure  Alcohol Lamp for 15 minutes at 121oC  70% Alcohol Page 2 of 3  PROCEDURE 1. Wipe the outer surface of the fruit with 70% alcohol and allow it to air dry completely. 2. Mark a section of the fruit peel, approximately 1 1⁄2 inches by 1 inch, for cutting. 3. Disinfect the knife or cutter thoroughly to maintain aseptic conditions. 4. Carefully cut the marked section of the fruit peel. 5. Gently press the inner (ventral) side of the fruit peel onto the agar surface in the petri dish. 6. Let the peel sit on the agar for 15 minutes, then carefully remove it from the petri dish. 7. In another petri dish, repeat the procedure 1-6 but allow the peel to remain on the agar for 30 minutes before removing it. 8. Wrap each plate securely with plastic wrap or parafilm and incubate the plates upside down (inverted). 9. Place the plates inside a shoebox or a similar container at room temperature for incubation. VI. MOLD CULTURE MATERIALS  Bread /Rice contaminated with Molds  Culture Media (PDA or SDA)  Marker  Plastic Wrap  Inoculating needle/Toothpick  Shoe Box  Alcohol lamp/Candle PROCEDURE 1. Open the Sabouraud Dextrose Agar or Potato Dextrose Agar plate. 2. Place the material contaminated with molds directly above the culture plate. 3. Gently tap the material to allow the spores to fall onto the agar surface. 4. Change your gloves to prevent cross-contamination. 5. Wrap the culture plate with plastic wrap or parafilm to secure it. 6. Store the wrapped plate inside a shoebox at room temperature for incubation. Page 3 of 3 MYCOLOGY AND VIROLOGY (LABORATORY) Quitaleg, Jenny Rose Maghuyop LAB PRELIM 2 SPECIMEN COLLECTION OUTLINE I. INTRODUCTION I. Introduction II. Laboratory Safety III. Specimen Collection, Handling, Transport  Fungi live as saprophytes in nature, and are capable of life cycle with or without human host.  Humans are typically resistant to fungal infections and become an accidental host by inhalation of spores or by introduction of fungi via tissue trauma.  Immunocompromised individuals exposed to non- pathogenic fungi could still develop infections. II. LABORATORY SAFETY Class II – should be used to reduce Biological personnel exposure to fungal Safety Cabinet elements – in mycology laboratories can be Petri dish hazardous due to the potential release of conidia or spores – high exposure to hazardous material due to the size of the petri dish – are recommended since the Screw-top tubes chance for the release of airborne conidia is less likely – low exposure hazardous material as tube has smaller opening than the petri dish  HEPA filter – high efficiency particulate air filter Page 1 of 4   Scrape using a blunt scalpel, tweezers, or III. SPECIMEN COLLECTION, HANDLING, bone curette. TRANSPORT  Scrape from the outer edge/active border of the surface lesion  All scraped lesions should be firmly rubbed with swab moistened in BHI Broth. – swabs are used to collect loose skins GENERAL PRINCIPLE  Place the specimen in a dry sterile container and store at room temperature  KOH wet mount - prepared with some of the scrapings; breaks down tissue to view fungal  Different types of specimens require different hyphae collection methods. – skin specimen needs to be KOH wet mount  Transport and process the specimen as soon as  Remaining material inoculated directly to possible. the agar. o delay in processing may compromised the specimen  Clean the skin with 70% isopropyl alcohol quality before sample collection  Specimen may be submitted as scrapings,  Specimen must be stored at suitable temperatures. cuttings, or complete nail  Collected and stored specimen must be processed  Sterile scissors are used to cut complete promptly and correctly. NAIL nails into smaller strips o when stored at room temperature for too long, it could  Pincer type nail clipper - used to clip cause overgrowth of bacterial contaminants through the entire thickness of the nail  Place the specimen in a dry sterile container  Hair, skin, or nails submitted for dermatophyte culture and store at room temperature are generally contaminated with bacteria, rapidly growing  Deeper scrapings for KOH preparation and fungi, or both. inoculate media o dermatophytes infects hair, skin and nails as they require keratin for growth  Primary isolation medium for dermatophyte culture should contain antimicrobial agents. SKIN, NAIL, HAIR, AND SCALP  Ideally, all specimens submitted for culture should be examined microscopically for fungi, provided they are of sufficient quantity.  Microscopic examinations prior to culture can be crucial WOOD'S LAMP for the early detection of fungi and early initiation of treatment for the patient.  If the specimen quantity is not sufficient, culture must take priority over the microscopic exam.  Developed by Robert Wood  Handheld device that emits long wave ultraviolet light at 340-365 nm SPECIMEN COLLECTION, HANDLING, TRANSPORT  Assists in identifying fungi-infected skin, nail, hair, and scalp  Produced little amount of UV light  No known negative effect SPECIMEN COLLECTION, HANDLING, TRANSPORT  For patients with suspected tinea or ringworm, any ointments or topical creams TINEA TINEA CAPITIS should be removed using an alcohol wipe VERSICOLOR prior to specimen collection. infection in the head  Scrape the infected scalp using blunt scalpel, tweezers, or bone curette reveal area of baldness – scrape the active border of the infected HAIR AND area most Microsporum Trichophyton SCALP – choose area with the loose skin Microsporum gypseum schoenleinii  Pull affected hair using sterile forceps species  Cut hairs close to the scalp using sterile scissors  Hairs are placed in a dry sterile container, yellowish-white blue-green dull yellow dull blue stored at room temperature or copper- color – urine container orange color – autoclaved petri dish – individually packed containers  Disinfect the skin and remove topical creams (if applied) with 70% isopropyl alcohol tinea – fungal infection based on the area of the body SKIN before sample collection infected  Soggy skin should be peeled away (such as in toe webs). Page 2 of 4  NON-PATHOGENIC BLOOD AND BONE MARROW  If fungal septicemia is suspected, the physician should HEALTHY SKIN DRY/DEHYDRATED LINT FROM inform the laboratory prior to specimen collection because SKIN CLOTHES special media are required for the optimal recovery of fungi. blue color purple color bright white and shiny 10ML BLOOD BONE MARROW ASPIRATES  aseptic collection with iodine and alcohol OTHER USES  smear with  smear with 1. Giemsa stain 1. Giemsa stain 2. Gram stain 3. Periodic Acid-Schiff  Disorders of pigmentation such as melasma and vitiligo (PAS) stain  Scabies and head lice REMAINING 3-5 ML OF REMAINING BONE MARROW  Corneal abrasion on the surface of the eye BLOOD ASPIRATES centrifuged centrifuged ↓ ↓ sediment must be cultured / sediment must be cultured / TO USE WOOD’S LAMP: processed processed 1. direct culture 1. direct culture 2. biphasic filter technique 2. biphasic filter technique 1. Clean the skin using water or alcohol. Remove make – up 3. membrane filter technique 3. membrane filter technique 4. lysis centrifugation 4. lysis centrifugation on the face. Remove deodorant or cream on the armpits. 2. Turn it on and wait for atleast 60 seconds before using it. 3. Use it in a dark room. Biphasic system: type of culture media that contains 4. Instruct the patient to close his eyes when it will be used broth (liquid phase) and agar (solid phase) may be used near the face. 5. Place the wood’s lamp 10 – 30 cm away from the skin of the infected area. 6. Examine area for few seconds only. first 4 days of incubation approx. 10 to 14 days of incubation most fungi are detected H. capsulatum is recovered SKIN, HAIR AND NAILS  Candida spp. is the most commonly recovered specie in the blood.  Optimal temperature for blood fungal culture is usually FUNGAL SCRAPINGS KIT FOR 30°C and must be maintained for 4 weeks. TRANSPORT CSF AND OTHER STERILE FLUIDS  Black collection cards that allows the better visualization of the collected specimen (skin, hair, nails) o petri dish or sterile container can also be used  3-5mL of CSF is optimal for fungal investigation, smaller  Size suitable for mail transport volumes are often received and should still be processed.  Transported with the test request and the swab  When processing is delayed, DO NOT refrigerate the o swab to clean the scraped area with BHI specimen.  Store at room temperature or incubate at 30°C.  Centrifuge the specimen before processing.  1 drop of concentrate must be used for India ink preparation or latex agglutination for Cryptococcus.  Remainder of the concentrate is inoculated onto culture media.  Media with antimicrobial agents should not be needed because CSF is normally sterile.  If >5 mL is submitted, the CSF may be filtered through a membrane filter and portions of the filter placed on media. Page 3 of 4  CSF in very invasive do not reject small amount >2 hours of delay short delays CSF amount increases when there is an infection may hinder fungal detection store the specimens at 4°C Centrifugation makes organisms concentrated at the (refrigerator temperature) bottom store in sterile specimen or petri dish TISSUE BIOPSIES URINE AND FECAL SPECIMEN  Perform by a physician  Ideally, tissue specimens should include both normal tissue and samples from the center and edge of the lesion.  First-voided morning urine specimen is preferred.  Tissue specimens must be moistened with saline or BHI  Midstream clean-catch or catheterized specimen Broth and must be transported to the laboratory minimizes the presence of normal flora. immediately.  Urine from collection bag, bed pans, or 24-hour collection  Suspicious area (purulent or discolored) are selected for are unacceptable. mincing and grinding prior to subsequent culture when  Specimen must be centrifuged before inoculation into large sections are submitted. culture media.  Urine samples must be processed as soon as possible. pus or necrosis is no pus or necrosis zygomycosis is present suspected >2 hours of delay at room short delays  inoculate  mince tissue  gently tease the temperature directly onto into small tissue apart isolation media pieces or grind and inoculate it may impede the detection of store the specimens at 4°C  prepare a soft tissues directly onto the some fungi smear for using a sterile isolation media microscopic glass tissue examination grinder  Urine, feces, and vaginal secretions are usually for bacteriologic culture but these specimens grow yeast that requires identification  Grinding process may destroy fragile fungal elements, particularly a zygomycete. PREDOMINANT CULTURE SITES FOR RECOVERY OF CAUSATIVE AGENTS RESPIRATORY SPECIMENS – saliva – sputum See last page. – bronchoalveolar lavage (BAL)  Many opportunistic fungi originate in the lungs due to Organisms may be recovered from multiple sites in inhalation of fungal spores. disseminated infections.  Specimen should be collected in the morning, as overnight fungal growth in the lungs increases the chances of isolating pathogenic fungi.  Deep cough shortly after arising in the morning or induce using a nebulizer. Collect in a sterile, screw cap container.  Patients should avoid eating before specimen collection.  24-hour samples are unacceptable because they can become overgrown with bacteria and fungal contaminants.  Lower respiratory tract specimens such as bronchial washings and sputum are often contaminated with normal flora. Interpreting results from poor-quality specimens may be challenging.  Throat specimens are collected by rolling a moist sterile swab over the affected area.  If Candida is suspected, the affected area should be scraped with a sterile tongue depressor.  All specimens should be sent in the laboratory immediately. Page 4 of 4  PREDOMINANT CULTURE SITES FOR RECOVERY OF CAUSATIVE AGENTS INFECTION RESPIRATORY BLOOD BONE MARROW TISSUE SKIN MUCUS Blastomycosis + + + Histoplasmosis + + + Coccidioidomycosis + + + Paracoccidioidomycosis + + + Sporotrichosis + + + Chromoblastomycosis + + Eumycotic mycetoma + + Phaeohyphomycosis + + MYCOLOGY AND VIROLOGY (LABORATORY) Quitaleg, Jenny Rose Maghuyop LAB PRELIM 3 SPECIMEN CULTURE AND PROCESSING OUTLINE I. INCUBATION I. Incubation II. Culture Media III. Specimen Processing FUNGI CULTURES IV. Laboratory Identification OF Significant Isolates  should be incubated at room temperature (25°C) or at 30°C (considered to be the highest room temperature)  fungi grow optimally BUT bacteria have slower growth rate at these temperature  if suspected as dimorphic fungi, it should also be incubated at 37°C DIMORPHIC FUNGI  fungi that can manifest both yeast and mold at 25°C at 37°C mold form yeast form EXAMINATION OF CULTURES  cultures are examined twice weekly and are maintained for 4 to 6 weeks (1 – 1 ½ months) INFORMATION RECORDED ABOUT THE ISOLATE INCLUDES  number of days until first visible growth  number of days required to see fruiting structures (mycelia)  whether mold or yeast forms are recovered  media on which the fungus is isolated  temperature at which growth occurs  morphology of the colonies Page 1 of 6  Mucorales (Mucor and Fonsecaea or Phialophora POTATO DEXTROSE AGAR (PDA) Rhizopus spp.) spp.  grow rapidly  slow growing organisms  may form aerial  might require 2 weeks mycelium within few or longer  Recommended for the isolation and enumeration of yeasts days and molds, and other fungi.  Stock cultures of certain dermatophytes can be maintained on PDA media.  PDA is also used for stimulating sporulation and differentiating typical varieties of dermatophytes based on their pigment production. II. CULTURE MEDIA  Solid phase PDA SUPPLEMENTED PDA SUPPLEMENTED Fungal culture media DO NOT o True WITH WITH share nutritional and CHLORTETRACYCLINE CHLORAMPHENICOL environmental needs that recommended for the recommended for the characterize bacteria microbial enumeration of selective cultivation of fungi ____________ are usually o Antimicrobials yeast and mold from from mixed samples incorporated with fungal media cosmetics Examples of antimicrobials o gentamicin mixed samples – o chloramphenicol bacteria and mold  o cycloheximide subculture inhibit bacterial growth o gentamicin and mostly used chloramphenicol inhibits bacteria and o Cycloheximide environmental fungi that are ISO 16212 contaminants _________ may be used for o petri dish or large test tubes  requires all manufactures to test their cosmetic products fungal media; both should be opened only in a BSC from mold contamination to prevent skin irritation and health issues PETRI DISH COMPONENTS ADVANTAGE DISADVANTAGE  Large surface area,  More prone to dehydration INGREDIENTS GRAMS/LITER easier to because of the prolonged Potato (infusion form) manipulate when incubation therefore must be nutrient source 200 making poured thicker than Dextrose preparations normal. type of sugar 20  Tape or parafilm or sealed source of energy in semipermeable bags to Agar 15 minimize dehydration and solidifying agent prevent spread of fungal 5.6±0.2 spores Final pH (at 25°C) 5.4  More hazardous 5.8 LARGE TUBES/TUBED MEDIA ADVANTAGE DISADVANTAGE SABOURAUD DEXTROSE AGAR (SDA)  Safer to handle  Smaller surface area. and less  Hard to get the mold susceptible to  A selective media for fungal culture and primarily used for drying. the isolation of dermatophytes, yeasts and various other pathogenic and non-pathogenic fungi.  Mycological analysis of food, cosmetics, personnel P hygiene, surfaces and air.  It is also used for the recovery and total counting of yeasts and molds in environmental monitoring. Page 2 of 6  SDA without antibiotics relied on low pH (5.6) for the  A more selective formula containing chloramphenicol inhibition of bacterial growth. and cycloheximide is used to recover pathogenic fungi  General type of culture media in mycology while inhibiting many bacteria and environmental fungi.  Can also be supplemented with antimicrobials  Also used in bacteriology COMPONENTS COMPONENTS INGREDIENTS GRAMS/LITER Peptones (meat and casein) 10 Dextrose monohydrate 40 source of energy Agar 15 Final pH (at 25°C) 5.6±0.2 5.4 5.8 POTATO DEXTROSE BROTH (PDB)  Recommended for the isolation and enumeration of yeasts PB50 & TB50 and molds.  For plate counts of yeasts and molds in the examination of basic BHI foods and dairy products. does not contain blood and antibacterials  Used for stimulating sporulation, for maintaining stock cultures of certain dermatophytes and for differentiation of Basic pH typical varieties of dermatophytes on the basis of pigment production. 7.2 7.6  Liquid phase COMPONENTS INGREDIENTS GRAMS/LITER Potato (infusion form) nutrient source 200 Dextrose type of sugar 20 source of energy 5.6±0.2 Final pH (at 25°C) 5.4 5.8 BRAIN HEART INFUSION AGAR (BHIA)  Enriched, non-selective media used for isolating and cultivating various bacteria, including yeasts and molds.  BHI Agar with (5 to 10%) defibrinated sheep blood is widely used for recovering dimorphic fungi like Histoplasma capsulatum and other pathogenic fungi such Photos from manufacturer as Coccidioides immitis. Page 3 of 6  TYPES LOCATION SUMMARY OF PRIMARY FUNGAL Tinea capitis scalp ringworm scalp CULTURE MEDIUM Tinea faciei face Tinea barbae beard Tinea corporis ringworm anywhere in the body Tinea manuum hands MEDIUM EXPECTED GROWTH RESULTS Tinea ungulum dermatophytes nails At 22°C onychomycosis, Tinea cruris jock itch groin SDA o Initial isolation of pathogens and Tinea pedis athlete’s foot foot saprobes o Dimorphic fungi may exhibit their mycelial phase SDA with o Saprobes are generally inhibited on antibiotics* this medium. o Dermatophytes and most of the fungi considered primary pathogens grow BHI o Initial isolation of pathogens and saprobes BHI with o Recovery of pathogenic fungi antibiotics o Dermatophytes not usually recovered Inhibitory mold o Initial isolation of pathogens except agar dermatophytes Cycloheximide o Primary recovery of dermatophytes At 37°C SDA o The yeast form of dimorphic fungi and other organisms grow o Dermatophytes grow poorly BHI with blood o Yeasts such as Cryptococcus grow well o The yeast form of Histoplasma IF CANDIDA IS SUSPECTED capsulatum takes up some of the heme pigment in the medium and becomes light tan, with a grainy, wrinkled texture SKIN SCRAPINGS DIRECT MICROSCOPY III. SPECIMEN PROCESSING  Prepare KOH wet mount and Calcofluor-stained mount  CULTURE SKIN, NAIL HAIR AND SCALP  Inoculate to Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35C. Maintain cultures for 4 weeks. IF TINEA IS SUSPECTED SKIN SWABS DIRECT MICROSCOPY DIRECT MICROSCOPY  Prepare KOH wet mount and Calcofluor-stained mount  Prepare Gram Stain in a sterilized glass slide CULTURE CULTURE  Inoculate specimen into two types of culture media containing cycloheximide  Moistened swab with BHI collected from lesions must be inoculated in Sabouraud's dextrose agar slope containing  Inoculate to Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide chloramphenicol and gentamicin, but NO cycloheximide  Incubate cultures at 26°C and maintain cultures for 4 and incubate at 35C. Maintain cultures for 4 weeks. weeks. Page 4 of 6  When secondary bacterial infection is suspected, the swab MEMBRANE FILTER TECHNIQUE should first be inoculated onto a blood agar plate, followed by the Sabouraud's agar containing the antibiotics and then placed into Brain Heart Infusion Broth. All cultures  Superior method compared to biphasic technique. should be incubated at 35C.  Specimens are treated sequentially with Triton-X and sodium carbonate solutions to lyse blood cells and then filtered by vacuum through a 0.45 um membrane.  The membrane is placed in in culture media. BLOOD AND BONE MARROW DIRECT CULTURE METHOD  Inoculate 0.5-1mL of buffy coat onto the surface of the culture media using a sterile inoculating loop  Incubate the plate aerobically at 30°C and maintain the cultures for 4 weeks.  Suitable for small, low volume laboratories where there are few requests for fungal blood cultures. LYSIS CENTRIFUGATION ISOLATOR SYSTEM BIPHASIC FILTER TECHNIQUE  Recommended by for patients with suspected fungal septicemia.  Enhance the recovery of fungi by using a biphasic bottle.  Utilizes a tube (requires 10mL of blood) that lyses  Contains a slant of brain heart infusion agar and 60-100 ml leukocytes and erythrocytes, inactivates plasma of BHI broth. complement, and certain antibiotics.  1:10 to 1:20 (blood to broth ratio) is recommended, a  Cell lysis releases microorganisms, which are minimum of 5.0 ml of blood is required. concentrated by centrifugation.  Cultures must be vented, checked, and tilted daily to allow  Concentrate is inoculated onto culture media and broth to flow over the agar surface. incubated at 30°C for 4 weeks.  Cultures should be incubated at 30°C and maintained for 4  Tube can withstand higher speed of centrifugation weeks.  Gram stain may be performed to the contents of the biphasic media to assist in detecting fungal elements. Page 5 of 6  CSF AND OTHER STERILE FLUIDS DIRECT MICROSCOPY  Centrifuge the specimen before processing.  Make wet mount preparations in KOH (l drop) and Gram  1 drop of concentrate must be used for India ink stained smears (l drop) of all suspicious areas. preparation or latex agglutination for Cryptococcus.  Periodic acid-Schiff Staining (PAS) may be necessary if  Resuspend the remaining concentrate/sediment in 1-2Ml the KOH preparation is unsatisfactory. of CSF before inoculating into culture media:  Sabouraud's dextrose agar with chloramphenicol and gentamicin. Incubate duplicate cultures at 26°C CULTURE and 35°C. Maintain for 4 weeks.  Brain heart infusion agar (BHIA) supplemented with 5% sheep blood. Incubate at 35°C. Maintain for Blood or pus present in the sample should be plated 4 weeks. directly onto media.  Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26C and TISSUE BIOPSIES 35C. Maintain cultures for 4 weeks.  Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 DIRECT MICROSCOPY weeks.  examination of tissue sections stained with the following URINE stains are essential:  Hematoxylin and Eosin Stain (H&E)  Grocott's Methenamine Silver Stain (GMS)  Centrifuge the urine for 10-15 minutes at 2000 rpm.  Periodic acid-Schiff Staining (PAS)  If necessary, decant the supernatant and pool the sediment. CULTURE DIRECT MICROSCOPY  Prepare a direct smear of the sediment in KOH  Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26C and 35C. Maintain cultures for 4 weeks.  Brain heart infusion agar (BHIA) supplemented with 5% CULTURE sheep blood and incubate at 35C. Maintain cultures for 4 weeks.  Inoculate 0.05-0.1 ml of the sediment onto Sabouraud's agar with gentamicin and chloramphenicol. RESPIRATORY SPECIMENS  Incubate duplicate cultures at 26C & 35C. Maintain cultures for 4 weeks.  Specimens that are not viscous can be inoculated onto media with a sterile pipette. IV. LABORATORY IDENTIFICATION OF  For viscous specimen: SIGNIFICANT ISOLATES  Dacron swab – used to inoculate viscous material such as thick tracheal aspirate  N-acetyl-L-cysteine – mucolytic agent such as may See last page. be used to digest the specimen before inoculation  Sterile Glass beads – shaking with 12-20 sterile glass beads and about 3-5ml of sterile distilled water, depending on the volume of the original specimen Page 6 of 6 MYCOLOGY AND VIROLOGY (LABORATORY) Quitaleg, Jenny Rose Maghuyop LAB PRELIM 4 FUNGAL MORPHOLOGY IN CULTURE MEDIA OUTLINE I. FUNGI CULTURE I. Fungi Culture II. Macroscopic Examination Of Cultures YEAST AND MOLD CULTURES III. Mold Colony Morphology IV. Mold Cultures V. Yeast Colony Morphology VI. Yeast Cultures Page 1 of 5  II. MACROSCOPIC EXAMINATION OF SIZE CULTURES  diameter of the colony  generally a visual comparison between genera or species GROSS MORPHOLOGIC TRAITS punctiform – tiny colonies – like a dot  must be recorded small ¼ of the petri dish medium ½ of the petri dish  surface big > ½ or whole petri dish COLOR pigment  reverse pigment on the pigment reverse side of FORM the colony or in the aerial mycelium can be noted but is not  basic shape of the colonies always helpful. TEXTURE rough  rapidly appear within 1-3 growing days GROWTH RATE organisms  intermediate appear within 5 growers to 9 days smooth  slow growers appear up to 2 weeks irregular fungi cultures must be maintained for 4 – 6 weeks before disposal mold form yeast form filamentous – commonly RT 37OC or around body seen than temperature rhizoid – dry rhizoid III. MOLD COLONY MORPHOLOGY COLONIAL CHARACTERISTICS OF THE ORGANISMS  helps the microbiologist to make an educated guess regarding the identification of the isolate  many of the following colony characteristics may vary among species and strains of the same genus  some colonies may be colored, some colonies are circular in shape, and others are irregular Page 2 of 5  ELEVATION SURFACE / TEXTURE  determined by tilting the culture plate and looking at the  appearance of the colony side of the colony smooth raised glistening rough wrinkled cottony – irregular strands convex flat velvety – like cottony – uniform layer – mouse fur umbilicate – depressed center granular – dots – concave - an – granules “innie” umbonate – raised or bulging center – convex - an “outie” OPACITY MARGIN transparent – clear translucent – not so clear – not totally opaque  the magnified shape of edge of a colony – some light can pass through entire – smooth opaque – no light can pass through – has solid color undulate Color (Pigmentation) surface pigment pigment on top of the colony filiform – strands reverse pigment pigment under the colony curled lobate Page 3 of 5  IV. MOLD CULTURES Trichosporon spp. Histoplasma capsulatum  Hair fragments implanted onto primary isolation media, like Sabouraud's dextrose agar  Colonies of Trichosporon spp. are white or yellowish to  Exhibits thermal dimorphism deep cream colored, smooth, wrinkled, velvety, dull  Mold form at soil or culture at temperatures around 30°C colonies  Yeast form in living tissue or in culture at 37°C V. YEAST COLONY MORPHOLOGY Trichophyton mentagrophytes  Yeasts can be easily mistaken for bacteria due to their similar colony morphology  They may appear as o small, o creamy or o white colonies  In gram stain are larger like sesame seeds than bacteria Candida species generally creamy white Candida krusei flat, dry colony morphology Cryptococcus species presumptively identified by their mucoid appearance, which is due to the production of abundant polysaccharide capsular material Rhodotorula rubra recognizable by its distinctive  Colonies are generally flat, white to cream in colour, with a orange-red pigmentation powdery to granular surface  Some cultures show central folding or develop raised central tufts or pleomorphic suede-like to downy areas  Reverse pigmentation is usually a yellow-brown to reddish-brown colour Page 4 of 5 VI. YEAST CULTURES Rhodotorula rubra Candida albicans Cryptococcus Candida krusei Page 5 of 5