Introductory Remarks

Transcription

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2 Introductory Remarks

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4 Introductory Remarks The Manual Sec6ons at beginning detail basic procedures, e.g., Gram staining, Microscopy, Oil immersion microscopy, Prepara6on of smears, Asep6c technique, Observa6on of colonies, Microbiological media, Abbrevia6ons and units Each prac6cal session is fully described with background informa6on and procedures Please try to read what you are doing before a laboratory session

5 Introductory Remarks Incuba6on of plates PLEASE ENSURE THAT YOU MARK PLATES WITH YOUR BENCH AND PLACE NUMBER OR ACCORDING TO SPECIFIC INSTRUCTIONS DO NOT PUT UNNECESSARY INFORMATION ON PLATES PLATES ARE INCUBATED INVERTED UNLESS SPECIFICALLY INSTRUCTED OTHERWISE

6 Yeasts

7 2. Diseases in Man caused by Yeasts Thrush Candida albicans, Candida spp. Oral thrush Intes6nal thrush Vaginal thrush Oesophageal thrush (AIDS pa6ents) Crytococcosis Cryptococcus neoformans An encapsulated yeast that causes meningi6s

8 Candida albicans and Oral Thrush Source: Atlas of Clinical Oral Pathology, 1999

9 Today s Objec6ves Part 1 Wet Mounts of yeasts (Sec6ons 2.1, 2.2, 2.3) Saccharomyces cerevisiae Schizosaccharomyces pombe Candida albicans Gram or Methylene Blue stains (Sec6ons 2.1, 2.2, 2.3) Saccharomyces cerevisiae Candida albicans Schizosaccharomyces pombe See Chlamydospores produced by Candida dubliniensis (2.4.1)

10 2.1. Saccharomyces cerevisiae

11 2.1 Saccharomyces cerevisiae Phase contrast microscopy (wet mount) Methylene blue stain

12 2.2 Schizosaccharomyces pombe Fission yeast

13 2.2. Candida albicans

14 Candida albicans Wet mount Buds arrowed Gram stain Buds arrowed

15 Candida spp. Dimorphic Budding growing as a yeast Pseudohypha growing as a fungus

16 Chlamydospores of Candida albicans (low power) These are terminal thickwalled survival spores which are not released.

17 Chlamydospores of Candida albicans Black arrows = Chlamydospores; White arrows= Suspensor cells

18 Chlamydospores of Candida dubliniensis

19 Today s Objec6ves Part 2 Isola6on of Candida spp. From your mouth (SecFon 2.5.1) Using phosphate buffered saline, rinse your mouth for 30 seconds Spit the rinse fluid back into container Inoculate 0.5 ml onto a Sabouraud Dextrose Agar plate Using sterile spreader, spread the inoculum over the SDA plate Allow plate to dry close to bunsen by leaving lid very slightly ajar and rota6ng occasionally Incubate at 30 C for 3 days.

20 3. Moulds or fungi

21 3. Ailments in Man caused by Moulds Allergy: Hay fever, allergic rhini6s Asthma: Alternaria, Cladosporium, Aspergillus Infec6on: Farmer s Lung Aspergillus species Mycotoxicosis: Aflatoxin produced by Aspergillus flavus or Ergot produced by Claviceps

22 Today s Objec6ves Part 3 Sporangia of moulds are used to par3ally ID them. Make Lactophenol Cocon Blue Prepara6ons of sporangia of moulds (Sec6on 3) Aspergillus Penicillium Mucor Set up a culture to cause Dimorphism of Mucor rouxii (4.1)

23 How to make a Lactophenol Cocon Blue (LCB) Prepara6on of Fungus

24 3.1. Aspergillus Aspergillus flavus, which produces aflatoxins, causing ear rot of corn

25 Fungal Dimorphism Fungal dimorphism can be defined as growth in either a mould form or as a yeast form This ability can be important in the pathogenesis of infec6ons caused by some fungi including Candida species

26 Examples of Dimorphic Fungal Pathogens Candida spp: Yeast cell is normal form; hyphal or pseudohyphal form is invasive causing infec6on 2. Histoplasma capsulatum: Hyphal form is saprophy6c; yeast form is parasi6c and causes disease. 3. Paracoccidioides brasiliensis: Hyphal or mycelial form is saprophy6c; yeast phase causes infec6ons

27 Candida albicans Yeast phase normal flora Hyphal phase - infection

28 Candida albicans in transifon Budding Yeasts Germ Tubes

29 Candida albicans Pseudohyphae with yeast type cells at hyphal junctions

30 Histoplasma capsulatum dimorphism Hyphae and sporangia (arrowed) Mycelial growth of hyphae on agar as saprophyte Hyphae and sporangia (arrowed) Yeast cells budding in tissue infection

31 Mucor dimorphic a genus of pin head moulds Pin head sporangia Sporangia full of sporangiospores

32 4.1 Fungal Dimorphism Mucor rouxii A Model System to study Factors known to affect dimorphism: Temperature (M. rouxii dimorphism is not temperature dependent) Reducing condi6ons anaerobiosis Oxygen tension CO2 concentra6on Nutrient concentra6on (may be host related)

33 Fungal Dimorphism in Mucor rouxii A Model System Filamentous mould with hyphae and sporangia (arrowed) Budding yeast

34 4.1 Mucor rouxii Dimorphism the Experiment Take molten SDA agar from water bath Inoculate with two or three loopfuls of sporangiospores (sporangia) of Mucor rouxii Mix molten agar to disperse sporangiospores through agar by rolling container between hands Pour inoculated molten agar into small plas6c beaker provided Allow agar to set Place beaker in container for incuba6on at C for 5 7 days

35 Today s Objec6ves Part 3 Microscopic Protozoa (Sec6on 6) demonstra6ons Amoeba proteus (6.1) (amoeboid) Paramecium caudatum (6.2) (Ciliate) Trypanosoma brucei (6.3) (Flagellate) sleeping sickness Entamoeba histolyfca cysts (6.4) enterifs, dysentery Plasmodium falciparum (6.5) malaria Cryptosporidium parvum (6.6) enterifs Giardia lamblia (6.7) enterifs

36 Protozoa Sec6on 6.2 Paramecium Ciliate

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38 6.3 Trypanosoma brucei

39 Plasmodium falciparum Malarial Parasite Life Cycle

40 Plasmodium falciparum ErythrocyFc Cycle

41 Plasmodium faciparum Rupturing Schizont Releasing Merozoites

42 Cryptosporidium parvum Complex Life Cycle

43 Unstained oocysts of Cryptosporidium parvum in faeces Four trophozoites emerging from oocyst

44 Giardia lamblia Life Cycle