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General Microbiology BIO 231 Prof. Dr. Adel Ahmed El-Mehalawy Microorganisms: Thery are minute organisms which cannot be seen by naked eye. They require microscope. Discovery of microorganisms: 1- Van Leevanhock: He discovered simple microscope (one lens) and examine a drop of water. 2- Robert Hooke: He discovered the compound microscope (more than one lens). 3- Muller: He examined microorganisms in rotted substances and water. 4- Spallinzi: He boiled water in a closed flask for one hour and he proved that this water did not contain microorganisms. 5- Schwman: He proved that yeast cells are the cause of alcoholic fermentation of sugar solution. 6- Pasteur: He discovered the pasteurization of milk by heating milk at 145of for 30 minutes. 7- Robert Koch: He isolated rod shaped bacteria which cause Anthrax from the blood of infected animals. 2 3 4 Sources of Microorganisms 1- water 2- Air 3- Soil 4- Milk 5- Food 6- Sewage. Methods of Sterilization A- Physical Methods: 1- By heat: a) dry heat by oven. b) Moist heat by autoclave. 2- Pasteurization: by boiling at 65oC for 30 minutes. 3- Radiation: a) by ultaviolet light. b) by ionizing radiation. 4- Filtration: to remove spores of microorganisms and not kill them. B- Chemical Methods: These chemicals are used to kill spores (bactericidal) or to prevent the growth of spores (bacteriostatic). These chemicals are used in case of substances which are sensitive to heat. 1- Salts: used in high concentrations. 2- Acids or alkalies: Make the pH of the medium unfavorable for groeth. 3- Alcohols: used at concentration 10%. Nutrient Medium 5 The nutrient medium used for growth of microorganisms must contain the following constituents: 1- Carbon and Nitrogen sources. 2- Phosphate and sulphate. 3- Microelements ans macroelements. Importance of Microorganisms I- In Industry: Some microorgansims are used in industry such as yeasts. They are used in alcoholic fermentation to produce alcohol and CO 2 from sugar solution. Yeasts are also used in baking (bread making) which help in raising the dough. Somw microorganisms are used to produce dyes. II- In Medicine: Microorganisms like bacteria and fungi cause serious diseases to man and animals. a- Bacteria: cause fever, pulmonary infection and intestinal infection. b- Fungi: cause a disease to the respiratory system (Aspergillosis). Cause diseases to the skin (dermatophytosis). III- In Agriculture: a- Bacteria: Cause diseases such as common scab and crown gall. b- Fungi Cause diseases such as powdery mildew, downy mildew, rusts, smuts, rotts and wilts. Useful Microorganism: 6 Some microorganisms are useful and are used to produce antibiotics such as Penicillin (Penicillium) and streptomycin (Streptomyces). Growth of Microorganisms Growth: It is the increase in number of cells. Rate of Growth: It is the difference in growth between two successive periods. Each organism has its typical growth curve, consisting of the following phases: 1- Lag Phase:It is the period of no growth. 2- Log Phase: It is the period of rapid growth. 3-Stationary Phase: It is the period in which the number of living cells equals the number of dead cells. 4- Death Phase: It is the period in which the number of dead cells is greater than the number of living cells. 7 Factors Affecting Growth I- Temperature: According to temperature requirement, microorganisms are classified into the following groups: ↓ ↓ Pyschrophiles Prefer low temp. (0-15oC). ↓ ↓ Mesophiles Prefer medium temp. (15-35oC). Thermophiles Prefer hogh temp. (35-55oC). 2- Aeration: Acoording to oxygen requirement, microoorganisms are classified into the following groups: ↓ ↓ Aeriobic ↓ Facultative Live only in presence can live in presence or of oxygen. absence of oxygen. ↓ Anaerobic live only in absence of oxygen. 3-pH- value: According to pH- value, microorganisms are classified into the following groups: 8 ↓ ↓ Acidophilic Prefer acidic medium (pH 4-5). ↓ ↓ Mesophilic prefer medium pH (pH 6.5-7). 9 Alkalophilic prefer alkaline medium (pH 8-9). Viruses Viruses differe from microorganisms in the following properties: 1- They contain one kind of nucleic acid, either DNA or RNA. 2- They are unable to reproduce outside living cells. Outside the living cells, it is called a virion or virus particle Structure: The virus particle consists of nucleic acid and a protein coat known as “capsis” (nucleocapsid). Sometimes, there is a membrane. Therefore, the virus may be naked or covered by a membrane. Capsid: Capsid consists of subunits called capsomers. The capsomers may be helical or cubical. 10 Classification of viruses: Viruses are classified according to their hosts into the following groups: ↓ ↓ ↓ Animal viruses Plant viruses They cause many diseases to They cause many diseases to animals and humans, such as plants such as tobacco mosaic Influenza, Tumor. Virus (TMV). Bacterial Viruses (Bacteriophage) They are viruses which use bacterial cells as hosts. Structure of bacteriophage: It consists of polyhedral head and a tail. 1-Head: It consists of capsomers and contain DNA. 2- Hollow stylus. 3- Basal part. 4- Adsorption spikes. Cultivation of viruses 1- Homogenization: It is the breakdown of tissue into small units. 2- Trypsinization: 11 It is the breakdown of small units into cells. This was carried out bybreaking the cementing material which join the cells, by proteolytic enzyme. 3- Washing: The cells are washed with salt solution. 4- The washed cells are cultivated in a suitable growth medium. Purification of viruses 1- Ultrafiltration: The suspension containing viruses passes through suitable filter which allow the virus particle to pass. 2- Ultra-centrifugation: The suspension containing viruses may be purified by two- step centrifugation process. The first step was carried out at low speed and the second step was carried out at high spped. 3- Precipitation: This process was carried out by using chemical agents like ethanol or ammonium sulphate. 12 Reproduction of viruses 1-Adsorption and penetration: The viral particle attach itself to the host cell and penetrate this host. 2- Nucleic acid replication: Each part form its complementary part. 3- Maturation: After replication, the host produces viral protein (capsomer). 4- Release:After maturation, the virus releases from the host cell. 13 Bacteria They are unicellular microorganisms ( the cell perform all the vital processes, such as reproduction, nutrition, etc.). Structure: 1- Cell wall: It protects the cell from the external environment, and it defines the shape of the cell. 2- Flagella: They are organs of motility. 3- Cytoplasm: It is the cell sap containing ribosomes. 4- Nucleus: It is primitive and consists of DNA. Shape: I- Spherical: They are also called cocci. a- Monococcus: Single spherical cell. b- Diplococcus: Single cell divides one time in one direction. c- Tetracoccus: Single cell divides two times in two directions. d- Sarcina: Single cell divides three times in three directions. 14 e- Streptococcus: Single cell divides several times in one direction. f- Staphylococcus: Single cell divides several times in several directions. II- Rod- shaped: They are also called bacilli. a- Monobacillus: Single rod-shaped cell. b- Diplobacillus: Single cell divides one time in one direction. c- Streptobacillus: Single cell divides several times in one direction. III- Curved rods: There are two types: a- Vivrio: which consists of one turn. b- Spirillum: which consists of more than one turn. Motility of bacteria: Some species of bacteria are motile by means of flagella. 1- Monopolar: with a single flagellum at one pole of the cell. 2- Bipolar: with a single flagellum at both poles of the cell. 3- Lophotrichous: with a single tuft of flagella at one pole of the cell. 15 4- Amphitrichous: with a tuft of flagella at both poles of the cell. 5- Peritrichous: with flagella over the entire surface of the cell. Staining of Bacteria 1- Make a bacterial suspension. 2- Put a drop of the suspension on a clean slide and spread it. 3- Fix on a gentle flame. 4- Put the dye (Crystal violet) for one minute. 5- Wash with tap water. 6- Put safranin for 30 second. 7- Wash with tap water. 8- Dry on a gentle flame. 9- Put a drop of oil and examine under microscope. Spores in bacteria ↓ ↓ ↓ Spore-former Non-spore former 16 Spore former: I- Position: ↓ ↓ ↓ Polar ↓ Subpolar Central II- Size: ↓ ↓ Smaller than ↓ ↓ like the cell the cell. larger than the cell. 17 Fungi General Characters: 1- They are heterotrophic (can not synthesize their own food). 2- They are eukaryotes (have a true nucleus). 3- They have cell walls. 4- They reproduce sexually and asexually by means of spores. Thallus: The body of the fungus is called thallus which consists of fine threads called “hyphae”, the hyphae interconnect to form what is called “mycelium”. The mycelium may be ↓ ↓ Acellular Which consists of multinucleate protoplasmic mass called “plasmodium”. ↓ Cellular which may be unicellular (single cell or holocarpic), or multicellular (many cells or eucarpic). The spore germinate giving germ tube which enlarge giving a ‘hypha”. Many hyphae interconnect to form a “mycelium”. 18 The hyphae may be ↓ ↓ ↓ Septate Non-septate (have septa) (have no septa) Structure of the fungal celll: 1- Fungal cell wall: It consists of polysaccharides, proteins and lipids. Function of cell wall: a- It determines the shape of the cell. b- It protects the cell from the external environment. 2- Septa:They are found in septate hyphae. They protect the cell from dry conditions. Thus, septate fungi are more tolerant to water stress than non- septate fungi. 19 3- Plasma membrane: It consists of lipids, proteins and sterols. It contains “ergosterol”. Thus, fungi are sensitive to fungicides due to presence of ergosterol. 4- Nucleus: It is a well defined and complete nucleus. It consists of a nuclear membrane, nuclear sap and a nucleolus containing RNA. 5- Secretory system It consists of Golgi apparatus, endoplasmic reticulum and vesicles. 6- Vacuoles: It has many functions: a- Storage of compounds. b- Accunulation of phosphate. c- Containing proteases for brakdown of proteins. d- Regulation of cellular pH. 20 Reproduction of fungi Asexual reproduction Types of asexual spores: ↓ ↓ Sporangiospores They are formed inside a sac Called “sporangia”. ↓ ___________________________________ ↓ ↓ Uniflagellate Biflagellate Have single flagellum have two flagella ↓ one anterior (tensil-type) ______________________ and one posterior (whiplash ↓ ↓ type). Anterior Posterior (tensil-type) (whiplash-type) ↓ Conidia They are formed free ________________________________________________________ ↓ ↓ Thallospores Conidiospores They are formed by transformation Of organs. ↓ They are formed as new elements ___________________________________ ↓ Arthrospores Formed by fragmentation ↓ Chlamydospores formed by swelling of cells ↓ ↓ Blastospores Phialospores Formed as buds. Formed on a flask structures called “phialides”. 21 22 Sexual Reproduction Fungi reproduce sexually on three successive steps: 1- Plasmogamy: It is the unionof two protoplasmic masses (male gamete and female gamete). 2- Karyogamy: It is the union of two different nuclei (one from male and the other from female). 3- Meiosis followed by mitosis: Meiosis is to reduce the diploid number of chromosomes (2 n) to the haploid number ( n ). Mitosis is to increase the haploid number of chromosomes. ♀ ♂ (n) (n) (2 n) ( n) (n) (n) (n) (n) 23 (n) Classification of fungi Kingdom: Protista Kingdom: Eumycota 1- The thallus may be cellular or 1- The thallus is cellular ( septate or non- acellular. Septate). 2- Spores are motile. 2- Spores are non- motile. Phylum __________________________________________________________ Chytridiomycota Hyphochytridiomycota With posterior Whiplash-type flagellum. with anterior tensil-typeFlagellum. Oomycota with one anterior and one Posterior flagella. Subkingdom ______________________________________________________________________________ Zygomyxcota Dikaryomycota 1- Mycelium is non-septate. 1- Mycelium is septate. 2- Karyogamy occurs directly. 2- Karyogamy is delayed. Phylum ______________________________________________________________________________ Ascomycota Basidiomycota Spores are formed inside a sac called (Ascus). Spores are formed on a flate structure Called (basidium). 24 Phylum: Oomycota General Characters: 1- Presence of non-septate mycelium. 2- Presence of motile zoospores. 3- Presence of biflagellate zoospores. Albugo portulacae: 1- It causes a disease called white rust. 2- It has non-septate mycelium. 3- It has a club-shaped sporangiospore. 4- The sporangiospore carry a chain of sporangia. 25 Plasmopara 1- causes a disease called downy mildew. 2- Presence of non-septate mycelium. Peronospora 1- causes a disease called downy mildew. 2- Presence of non-septate Mycelium. Bremia 1- causes a disease called downy mildew. 2- Presence of nonSeptate mycelium. 3- Presence of mono- 3- Presence of dichoto- 3- Presence of dichoto- podially branched mously branched mously branched sporangiophore. Sporangiophore. Sporangiophores. 4- Presence of acute tips. 4- Presence of discshaped tips. 26 Subkingdom: Zygomycota General Characters: 1- Thallus is cellular. 2- Hyphae are non-septate. 3- Spores are non-motile. 4- Karyogamy occurs directly. Rhizopus Mucor 1- Saprophytic. 1- Saprophytic. 2- Presence of non-septate 2- Presence of non- mycelium. 3- Presence of three sporangio- septate mycelium. 3- Presence of one sporangiophore. Phores. 27 Subkingdom: Dikaryomycota General Characters: 1- Thallus is cellular 2- Hyphae are septate. 3- Spores are non-motile. 4- Karyogamy is delayed. Phylum: Ascomycota General Characters: 1- Thallus is cellular. 2- Hyphae are septate. 3- Spores are non motile. 4- Karyogamy is delayed. 5- Spores are formed inside a sac called “ascus”, each ascus contain 4-8 ascospores. 28 Leveillula Erysiphe Sphaerotheca 1- Cause a disease called 1- cause a disease called 1- cause a disease called powdery mildew. powdery mildew. powdery mildew. 2- Presence of septate 2- presence of septate mycelium. mycelium. 2- Presence of septate mycelium. 3- Presence of single 3- Presence of single 3- Presence of single conidiophore. conidiophore. Conidiophore. 4- Conidiophore carry 4- Conidiophore carry 4- Conidiophore carry single conidium. achain of conidia. a chain of conidia. 5- Conidiophore is short. 29 5- Conidiophore is long. Aspergillus Penicillium 1- Saprophyte 1- Saprophyte 2- Presence of septate 2- Presence of septate mycelium. mycelium. 3- Presence of unbranched 3- Presence of branched conidiophore. conidiophore. 4- Conidiophore ends with 4- Each branch ends with a vesicle carrying phialides. a phialide. 5- Phialide carry a chain of 5- Phialide carry a chain conidia. conidia. 30 Phylum: Basidiomycota General Characters: 1- Thallus is cellular. 2- Hyphae are septate. 3- Spores are non-motile. 4- Karyogamy is delayed. 5- Spores are formed on a flate structure called basidium. Puccinia graminis General Characters: 1- Causes a disease called black stem rust. 2- Mycelium is septate. 3- During its life cycle, it produces 5 types of spores: a- Basidiospores: Unicellular, Uninucleate, non-stalked and minute in size. b- Pycniospores: Unicellular, uninucleate, non-stalked formed inside a pycnium. c- Aeciospores: Unicellular, binucleate, non-stalked formed inside aecium. d- Urediospores: Unicellular, binucleate, stalked and double-walled. e- Teliospores: Bicellular, stalked and thick-walled. 31 32