Microbiology Week 11 and Experiments

        Assalamualaikum and Good morning.... How r you everyone? I hope everyone will be fine as well. 😊😊😊 This is eleventh week of my microbiology class and and my basic technique of microbiology class. In basic technique of microbiology class, we did our experiment 16 which is Physical factors: Atmospheric Oxygen Requirement and experiment 17 which is Techniques for the Cultivation of Anaerobic Microorganisms. In microbiology, for the whole week we don't have our microbiology class because Dr Wan went to Sydney. So that, on that day Dr Wan asked us to go to Institute Bioscience to visit and learning about electron microscope which is scanning electron microscope and transmission electron microscope.

Basic techniques of microbiology

        Dr Fairol give some briefing and explanation about the experiment 16 and experiment 17. On tuesday, we did the experiment 16 using nutrient broth cultures, Sabouraud broth cultures and thioglycollate broth culture and experiment 17 using nutrient broth cultures and thioglycollate broth cultures. Encik Hussain also give some demo and explanation about the both experiment because this both experiments are difficult and need more careful and concentration while we did the experiments.

Physical Factors: Atmospheric Oxygen Requirement

    Microorganisms exhibit great diversity in their ability to use free oxygen for cellular respiration. These variations in oxygen requirements reflect differences in biooxidative enzyme systems present in the various species. Microorganisms can be classified into one of five major groups according to their oxygen needs:

    Aerobes requires the presence of atmospheric oxygen for growth. Their enzyme system necessitates use of oxygen as the final hydrogen (electron) acceptor in the complete oxidative degradation of high-energy molecules, such as glucose. Microaerophiles require the absence of free oxygen for growth because their oxidative enzyme system requires the presence of molecules other than oxygen to act as the final hydrogen (electron) acceptor. In these organisms, as in aerobes, the presence of atmospheric oxygen result in the formation of toxic metabolic end products, such as superoxide, oxygen , a free radical of oxygen. Aerotolerant anaerobes are fermentative organisms, and therefore they do not use oxygen as a final electron acceptor. Unlike the obligate anaerobes, they produce catalase and/or superoxide dismutase, and thus they are not killed by the presence of oxygen. Facultative anaerobes can grow in the presence or absence of free oxygen. They preferentially use oxygen for aerobic respiration. However, in an oxygen-poor environment, cellular respiration may occur anaerobically, utilizing such compunds as nitrates or sulfates as final hydrogen acceptors, or via a fermentative pathway.

    The oxygen needs of microroganisms can be determined by nothing their growth distributions following a shake-tube inoculation. This procedure requires introduction of the inoculum into a melted agar medium, shaking of the test tube to disperse the microorganisms throughout the agar, and rapid solidification of the medium to ensure that the cells remain dispersed.

Techniques for the Cultivation of Anaerobic Microorganisms

      Microorganisms differ in their abilities to use oxygen for cellular respiration. Respiration involves the oxidation of substrates for energy necessary to life. A substrate is oxidized when it loses hydrogen ion and its electron. Since the loses hydrogen ion and its electron cannot remain free in the cell, it must immediately be picked up by an electron acceptor, which becomes reduced. Therefore, reduction means gaining the loses hydrogen ion and its electron. These are termed oxidation-reduction reactions. Some microorganisms have enzyme systems in which oxygen can serve as an electron acceptor, thereby being reduced to water. This discussion is limited to cultivation of the strict anaerobes, which cannot be cultivated in the presence of atmospheric oxygen. The procedure is somewhat more difficult because it involves sophisticated equipment and media enriched with substances that lower the redox potential. This experiment uses fluid thioglycollate medium and the GasPak anaerobic system.
                                         Experimet 16: nutrient broth cultures of Enterococcus faecalis

Experiment 16 : Sabouraud broth cultures of Saccharomyces cerevisiae

Experiment 17: nutrient agar plate of aerobic of Bacillus cereus and Escherichia coli

Experiment 17: thioglycollate broth culture of Clostridium sporogenes

Microbiology
Electron microscope

       An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects. A scanning transmission electron microscope has achieved better than 50 pm resolution in annular dark-field imaging mode[1] and magnifications of up to about 10,000,000x whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x. Electron microscopes have electron optical lens systems that are analogous to the glass lenses of an optical light microscope. Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used for quality control and failure analysis. Modern electron microscopes produce electron micrographs using specialized digital cameras and frame grabbers to capture the image.
The Scanning Electron Microscope (SEM) produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning). When the electron beam interacts with the specimen, it loses energy by a variety of mechanisms. The lost energy is converted into alternative forms such as heat, emission of low-energy secondary electrons and high-energy backscattered electrons, light emission (cathodoluminescence) or X-ray emission, all of which provide signals carrying information about the properties of the specimen surface, such as its topography and composition. The image displayed by an SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated. In the SEM image of an ant shown below and to the right, the image was constructed from signals produced by a secondary electron detector, the normal or conventional imaging mode in most SEMs. Generally, the image resolution of an SEM is lower than that of a Transmission Electron Microscope (TEM). However, because the SEM images the surface of a sample rather than its interior, the electrons do not have to travel through the sample. This reduces the need for extensive sample preparation to thin the specimen to electron transparency. The SEM is able to image bulk samples that can fit on its stage and still be maneuvered, including a height less than the working distance being used, often 4 millimeters for high resolution images. The SEM also has a great depth of field, and so can produce images that are good representations of the three-dimensional surface shape of the sample. Another advantage of SEMs comes with environmental scanning electron microscopes (ESEM) that can produce images of good quality and resolution with hydrated samples or in low, rather than high, vacuum or under chamber gases. This facilitates imaging unfixed biological samples that are unstable in the high vacuum of conventional electron microscopes.
   The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron beam to illuminate the specimen and create an image. The electron beam is produced by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the "image") may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera. The image detected by the digital camera may be displayed on a monitor or computer. The resolution of TEMs is limited primarily by spherical aberration, but a new generation of aberration correctors have been able to partially overcome spherical aberration to increase resolution. Hardware correction of spherical aberration for the high-resolution transmission electron microscopy (HRTEM) has allowed the production of images with resolution below 0.5 angstrom (50 picometres) and magnifications above 50 million times. The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research and development.

Microbiology Week 10 and Experiments

        Assalamualaikum and Good night.... How r you everyone? I hope everyone will be fine as well. 😊😊😊 This is tenth week of my microbiology class and and my basic technique of microbiology class. In basic technique of microbiology class, we did our experiment 14 which is Physical factors: Temperature and experiment 15 which is Physical factors: pH of the Extracellular Environment. In microbiology, On wednesday, First Dr Wan asked to do concept mindmap about protists. After that, Dr Wan taught us about the topic of Protists. 😇😇😇 On friday, We all having our test 2 exam.

   Basic techniques of microbiology

     Dr Fairol give some briefing and explanation about the experiment 14 and experiment 15.On tuesday, we did the experiment 14 using agar plate and Sabouraud broth tubes containing inverted Durham tubes and 15 using soy broth (TSB) tubes. On wednesday, we observe the agar plate and the absorbance reading of spectrometer.

Experiment 14: Physical Factors: Temperature

Microbial growth is directly dependent on how temperature effects cellular enzymes. With increasing temperatures, enzyme activity increases until the three-dimensional configuration of these molecules is lost because of denaturation of their protein structure. As the temperature is lowered toward the freezing point, enzyme inactivation occurs and cellular metabolism gradually diminishes. At 0°C, biochemical reactions cease in most cells. Bacteria, as a group of living organisms, are capable of growth within an overall temperature range of minus 5°C to 80°C. Each species, however, requires a narrower range that is determined by the heat sensitivity of its enzyme systems.

Minimum growth temperature:
The lowest temperature at which growth will occur. Below this temperature, enzyme activity is inhibited and the cells are metabolically inactive so that growth is negligible or absent.

Maximum growth temperature:
The highest temperature at which growth will occur. Above this temperature, most cell enzymes are destroyed and the organism dies.

Optimum growth temperature:
The temperature at which the rate of reproduction is most rapid; however, it is not necessarily optimum or ideal for all enzymatic activities of the cell.

Psychrophiles
Bacterial species that will grow within a temperature range of -5°C to 20°C. The distinguishing characteristics of all psychrophiles is that they will grow between 0 degree celsius and 5°C.

Mesophiles
Bacterial species that will grow within a temperature range of 20°C to 45°C . The distinguishing characteristics of all mesophiles are their ability to grow at human body temperature (37°C) and their inability to grow at temperatures above 45°C. Mesophiles with optimum growth temperature between 20°C and 30°C are plant saprophytes. Mesophiles with optimum growth temperature between 35°C to 40°C are organisms that prefer to grow in the bodies of warm-blooded hosts.

Thermophiles
Bacterial species that will grow at 35°C and above. Facultative thermophiles is organisms that will grow at 37°C, with an optimum growth temperature of 45°C to 60°C. Obligate thermophiles is organisms that will grow only at temperatures above 50°C, with optimum growth temperatures above 60°C.

Sabouraud broth tubes containing inverted Durham tubes of Saccharomyces cerevisiae in 20°C

Trypticase soy agar plates of Escherichia coli, Bacillus stearothermophiles, Pseudomonas aeruginosa and Serratia marcescens at 4°C

Experiment 15: Physical Factors: pH of the extracellular environment

Growth and survival of microorganisms are greatly influenced by the pH of the environment, and all bacteria and other microorganisms differ as to their requirements. Based on their optimal pH, microorganisms may be classified as acidophiles, neutrophiles or alkalophines. Each species has the ability to grow within a specific pH range; the range may be broad or limited, with the most rapid growth occurring within a narrow optimum range. These specific pH needs reflect the organisms' adaptations to their natural environment. For example, enteric bacteria are capable of survival within a broad pH range, which is characteristics of their natural habitat, the digestive system. Bacterial blood parasites, on the other hand, can tolerate only a narrow range; the pH of the circulatory system remains fairly constant at approximately 7.4.



Microbiology Week 10 (Protists)

Nutrition in Protists
• Protozoa are chemoheterotrophic protists
• Photoautotrophic protists
• Mixotrophic protists

Protist Morphology
- plasma membrane structure similar to multicellular plants/animals
- cytoplasm sometimes subdivided into outer gelatinous ectoplasm just underneath plasma membrane and inner fluid region termed endoplasm
- pellicle structure provides support
- vacuoles commonly present in protists
- energy production
- cilia/flagella may be present for motility/feeding

Protozoa
Different from prokaryotes-- relatively larger and
eukaryotic nature
Different from algae -- no chlorophyll
Different from yeast and fungi--by their motility and
lack of cell wall
Different from slime molds--lack of fruiting body
Unicellular, eukaryotic animal-like protists
Predatory or parasitic
Some are pathogenic
Aerobic, anaerobic chemoheterotrophic organism
Colorless and motile
Reproduction
-Asexually by fission, budding, multiple fission (schizogony)
-Sexual reproduction is conjugation/produce gametes.
Found in water and soil.
Some are part of the normal flora of animals

Nutrition of protozoa
Mostly aerobic heterotrophs, some are capable of anaerobic growth.
Dinoflagellates and Euglenoids are capable of photosynthesis
Obtain food through:
-Ingestion/swallowing of particulate or whole bacteria via gullet/cytostome
-Pinocytosis - fluid sucked into a channel
-Phagocytosis - surrounding the food particle with their flexible cell membrane
-Absorption - through plasma membrane

Archaezoa
• flagellated
• two or more flagella
• move in whiplike manner
• free-living
• parasitic/pathogenic
• Freshwater
• Trypanosoma gambiense
• African sleeping sickness-in human
• Lives and grow in the blood stream– inflammation of the brain and spinal cord

Rhizopoda
• Amoebas
• move by pseudopods- ameboid movement
• phagocytosis- obtain food
• habitat-freshwater, marine
• cause amoebic dysentery – human
• Transmitted from person to person in the cyst form – fecal contamination
• eg. Amoeba proteus, Entameoba histolytica

CILIOPHORA (ciliates)
• possess cilia
• 2 kind of nuclei
• Micronucleus - inheritance and sexual reproduction
• Macronucleus - production of mRNA
• best known - Paramecium
• presence of gullet (mouth)/cystostome - ingesting particulate material
• habitat- freshwater, marine
• Balantidium coli - human parasite - Dysentery

APICOMPLEXA
• not motile in mature forms.
• obligate parasites.
• food are absorbed through the outer wall.
• complex life cycle - transmission between several hosts.
• primarily animal parasites, insects.
• Plasmodium vivax - causing Malaria.
• Toxoplasma - causing toxoplasmosis.

Algae 
– They have a simple morphological construction
– Naked reproduction structures - asexual (all) and sexual (some)
– Mostly photoautotropic
– Contain chlorophyll a and other pigment
– Requires water
– Found virtually in any habitat on earth
– Classified according to thei rRNA sequences, structures, pigments and other qualities.

Habitat
 • aquatic habitat- fresh water, marine and brackish
 • moist soils and artificial aquatic habitat (fish tanks, pool)
 • few in dry soils
 • acidic habitat

Motility of Algae
 • If motile, due to flagella:
 • Single flagella—Euglena
 • Two or 4 polar flagella- Chlorophyta
 • Two flagella of different length and point of insertion—Dinoflagellates
 • Most cases, non-motile in vegetative stage and form mo)le gametes only during sexual  reproduction

Distribution of Algae
- Planktonic
- Benthic
- Neustonic

Chlorophyta
• Green algae
• Cellulose cell walls
• Unicellular or multicellular
• Chlorophyll a and b
• Store glucose polymer
• Gave rise to terrestrial plants

Rhodophyta
• reddish colour
• delicately branch thalli
• Chlorophylls a and d, phycocyanin, phycoerythrin
• red pigment absorb blue light
• mostly multicellular
• cell wall- cellulose/agar
• sexual reproduction
• storage material- glucose polymer
• habitat- marine (greater ocean depths)

Phaeophyta
• brownish colour
• macroscopic
• Chlorophylls a and c, xanthophylls
• multicellular
• cell wall- cellulose/algin
• sexual reproduction
• storage material- carbohydrate
• habitat- marine (coastal water)

Chrysophyta  (Diatoms-Bacillariophyta)
• golden-brown algae
• produce domoic acid- domoic acid intoxication
• Chlorophylls a, and c,
• unicellular
• cell wall- peptin and silica
• sexual reproduction
• storage material- oil
• habitat- fresh water, marine, soil
• Important in global carbon cycling – marine planktonic diatoms produce 40–50% of organic ocean carbon

Phyrrophyta  (Dinoflagellata)
 • unicellular plankton
 • brownish
 • 2 flagella in perpendicular opposing grooves
 • Some produce neurotoxins
 • can cause ‘red tides’- gives ocean a deep red colour
 • Chlorophylls a and c
 • cell wall- cellulose
 • storage material- starch
 • habitat- freshwater, marine

Euglenophyta (also considered with the protozoa)
 • unicellular flagellated
 • green colour
 • Chlorophylls a and b, carotene
 • can spontaneously lost chlorophyll (dark)- heterotrophic organism
 • cell wall- none
 • rigid plasma membrane-pellicle
 • no sexual reproduc0on
 • storage material- glucose polymer
 • habitat- freshwater, a few marine

Slime Molds

- Resemble fungi in appearance and life-style

 - Different in cellular organiza)on, reproduc)on and life cycles

 - Three divisions

 a) Myxomycota (Plasmodial slime molds)

 b) Acrasiomycota (Cellular slime molds)

 c) Peronosporomycetes (Water molds)

Myxomycota 

- plasmodial (acellular) slime molds

- glistening, viscous masses of slime 

- saprophytes

- multinucleated

- motile amoeboid mass called plasmodium (lack of cell wall)

- phagocytosized dead material 

Acrasiomycota 

 - individual amoeboid cells (unicellular)

 - feed phagocytically

 - plentiful of food, divide by mitosis and cytokinesis  

 - Cellular slime molds

 – great interest to cell and developmental biologists

 – provide a comparatively simple and easily manipulated system

 – for understanding how cells interact to generate a multicellular organism

 Peronosporomycetes 

 • “Egg fungi” formerly called oomycetes are diploid and no chitin in cell wall

 • Some grow in cottony masses on dead algae and animals

 • Some parasites of fish gills

 • Plant diseases include blue mold on tobacco and Irish potato blight

Distribution and functions of molds:

 - Moist terrestrial habitats e.g. soil, decaying wood, dung and etc.

 - Engulf bacteria (as predator)

 - As decomposer and consumer in the ecosystem

 - Recycling of nutrients (regenerate consumer’s waste, allowing plants to reuse nutrients)

 - Cause diseases in plants e.g. tobacco plants, potatoes, grapes

           

Microbiology Week 9 and experiments

        Assalamualaikum and Good night.... Hw r you everyone? I hope everyone will be fine as well. 😊😊😊 This is ninth week of my microbiology class and and my basic technique of microbiology class. In basic technique of microbiology class, we did our experiment 12 which is nutritional requirements: Media for the routine cultivation of bacteria and experiment 13 which is Use of differential, selective, and enriched media.  In microbiology, First Dr Wan asked us do to the remaining questions by other groups. After that, Dr Wan taught us about the topic of Fungi. Later on, Dr Wan was invited our seniors of microbiology student to make some discussion with us about the Fungi. 😇😇😇

Basic techniques of microbiology

     Dr Fairol give some briefing and explanation about the experiment 12 and experiment 13. My first thought about this experiments especially experiment 12 is I cannot understand the steps after Encik Zainuddin gave some demo then only I can understand so far. I asked my demo for my further understanding. On wednesday, as usual we observe the agar plate and record the reading of optical density using spectrophotometer.

Experiment 12: Nutritional requirements: Media for the routine cultivation of bacteria

    To satisfy the diverse nutritional needs of bacteria, bacteriologists employ two major categories of media for routine cultivation. Chemically Defined Media are composed of known quantities of chemically pure, specific organic and/or inorganic compounds. Their use requires knowledge of the organism's specific nutritional needs. Chemical defined media which are inorganic synthetic broth and glucose salts broth. Inorganic synthetic broth is inorganic medium is prepared by incorporating the following salts per 1000 ml of water which are sodium chloride is 5 g, magnesium sulfate is 0.2 g, ammonium dihydrogen phosphate is 1.0 g, dipotassium hydrogen phosphate is 1.0 g and atmospheric carbon doixide. Glucose salts broth is composed of salts incorporated into the inorganic synthetic broth medium plus glucose, 5 g per liter, which serves as the sole organic carbon source. Complex media is the exact chemical composition of these media is not known. They are made of extracts of plant and animal tissue and are variable in their chemical composition. Most contain abundant amino acids, sugars, vitamins, and minerals; however the quantities of these constituents are not known. They are capable of supporting the growth of most heterotrophs. Complex media which is nutrient broth and yeast extract broth. Nutrient broth is basic complex medium is prepared by incorporating the following ingredients per 1000 ml of distilled water which is peptone is 5.0 g and beef extract is 3.0 g. Yeast extract broth is composed of the basic artificial medium ingredients used in the nutrient broth plus yeast extract, 5 g per liter, which is a rich source of vitamin B and provides additional organic nitrogen and carbon compunds. Measuring turbidity, we can evaluate the abilities of media to support the growth of different species of bacteria and nutritional needs of the bacteria. We can observe the amounts of growth, measured by turbidity, present in each culture following incubation.

Experiment 13: Use of Differential, selective and enriched media

     Isolation of bacteria is accomplished by growing ("culturing") them on the surface of solid nutrient media. Such a medium normally consists of a mixture of protein digests (peptone, tryptone) and inorganic salts, hardened by the addition of 1.5% agar. Examples of standard general purpose media that will support the growth of a wide variety of bacteria include nutrient agar, tryptic soy agar, and brain heart infusion agar. A medium may be enriched, by the addition of blood or serum. Examples of enriched media include sheep blood agar and chocolate (heated blood) agar. Selective media contain ingredients that inhibit the growth of some organisms but allow others to grow. Selective media which is phenylethyl alcohol agar, crystal violet agar and 7.5% sodium chloride agar. For example, mannitol salt agar contains a high concentration of sodium chloride that inhibits the growth of most organisms but permits staphylococci to grow. Differential media contain compounds that allow groups of microorganisms to be visually distinguished by the appearance of the colony or the surrounding media. Differential/Selective media which is mannitol salt agar, MacConkey agar and eosin-methylene blue agar. Enriched media are media that have been supplemented with highly nutritious materials, such as blood, serum or yeast extract, for the purpose of cultivating fastidious organisms. Blood agar is one type of enriched media, allowing bacteria to be distinguished by the type of hemolysis produced. Hemolysis which is gamma hemolysis, alpha hemolysis and beta hemolysis.

 Eosin-methylene blue agar of Escherichia coli, Enterobacter aerogenes, Salmonella typhimurium and Staphylococcus aureus

Microbiology week 9 (Fungi)

Characteristics of fungi
- multicellular and multinucleated (except
yeast) yeast is unicellular
- spore-bearing organisms
- chemoorganoheterotrophs with
absorptive metabolism
- saprophytes - osmotrophy
- no chlorophyll
- rigid cell wall chitin
- primary storage of p/s: glycogen
-Reproduce sexually and asexually
- are stationary organisms disperse by wind,
water and animals

Fungal Distribution and Importance
-Primarily terrestrial, few aquatic
-Primarily terrestrial
-Many are pathogenic in plants or animals
-Industrial importance
-Research use

Fungal Structure
• Cell walls composed of chitin polysaccharide
 • Single-celled microscopic fungi = yeasts
 • Body/vegetative structure of a fungus = thallus (pl. thalli) – multicellular fungi are called molds – thallus consists of long, branched hyphae filaments tangled into a mycelium mass

Feeding forms of fungi
• Saprobic heterotrophs – feed on dead or decaying organic matter
• Parasitic heterotrophs – feeding on living host at cost to the host
• Mutualistic heterotrophs – feeding of or with a living host without damaging the host, both host and fungus benefit

Fungal Reproduction
Asexual reproduction 
– Parent cell undergoes mitosis to form daughter cells
– Mitosis in vegetative cells may be concurrent with budding to produce a daughter cell
– May proceed through a spore form
Sexual reproduction
 – Involves fusion of compatible nuclei
    • Homothallic: Sexually-compatible gametes are formed on the same mycelium (self-fertilizing)
    • Heterothallic: Require outcrossing between different, yet compatible mycelia
– A dikaryotic stage can exist temporarily prior to fusion of two haploid nuclei

Sexual spores (Types)
 • Zygospore - large spore enclosed in a thick wall.
 • Ascospore - produce in a sac like structure called ascus.
 • Basidiospore - formed externally at a base of basidium.

Life cycle
• Filamentous fungi can reproduce asexually by fragmentation
• Sexual and asexual reproduction- occurs by formation of spores
• Fungi are classified and identified by spore type
• Fungus spores and bacterial spore are different
• Spores are formed from the aerial mycelium

TYPES OF FUNGI
 • Chytridiomycota
• Zygomycota
• Ascomycomycota
• Basidiomycota
• Microsporidia

Chrytridiomycota (Anaerobic Rumen Fungi)
 • Neocallimastigales
 • Obligate anaerobes
 • Decompose cellulose
 • Breakdown lignin deposits into smaller pieces

Phylum Zygomycota
• Terrestrial – decomposers – mutualists: mycorrhizae
• Form coenocytic hyphae containing numerous haploid nuclei
• Form zygosporangia – dikaryotic, resistant stage
• Bread Mold, Rhizopus stolonifer
• foods, antibiotics and other drugs, meat tenderizer, and food coloring

Phylum Ascomycota
-Sac fungi
-red, brown, and blue-green molds cause food spoilage
-some are human and plant pathogens
-some yeasts and truffles are edible
-some used as research tools

Basidiomycota
• Basidiomycetes (club fungi) – examples include rusts, shelf fungi, puffballs, toadstools, mushrooms – sexual reproduction form basidium
• basidiospores are released at maturity

Human Impact Basidiomycota
 • Decomposers
 • Edible and non-edible mushrooms – toxins are poisons and hallucinogenic
 • Pathogens of humans, other animals, and plants

Microsporidia
• Obligate intracellular fungal parasites that infect insects, fish, and humans – Aquatic birds are common hosts and contribute to large numbers of spores in environment
• Transitional form is a spore structure capable of surviving outside the host
• Structurally similar to ‘classic’ fungi – contain chitin, trehalose, and mitosomes – however, lack mitochondria, peroxisomes and centrioles – unique morphology is polar tube essential for host invasion

Mycorrhizae
- Mutualistic association (plant root and fungi)
- Benefits for plants: increase surface area and  increase the growth potential
- Benefit for fungi: feeding from tissues of the plant
 - Mycorrhizal fungi : truffles, Auricularia

Lichens
• Mutualistic relationship between algae (or cyanobacteria) and fungi
• The algae produces food and the fungus portion provides protection, water, and minerals.
• Lichens may grow in extreme conditions (ex. Arctic areas too cold for most plants)
• Lichens are sensitive to environmental toxins, so they serve as indicators of the ecological health of an area.

Ecological impact of fungi
•Fungi as decomposers
•Fungi to modify habitat
•Fungi as spoilers
•Fungi to improve plant growth
•Fungi as food
•Fungi as pathogens

Yeast
Non filamentous
• Unicellular
• Typically spherical or oval
• Fission yeast – divide evenly to produce new cells
• Budding yeast – divide unevenly
• Yeast are capable of facultative anaerobic growth
• Baking
• Fermenting alcoholic beverages – Beer, wine, distilled spirits
• Model organism in cell biology research
• Microbial fuel cells
• Opportunistic pathogens
• Saccharomyces carlsbergensis (pastorianus)
• Brettanomyces (wild yeast)
• Bioremediation
• Yarrowia lipolytica is known to degrade palm oil mill effluent, TNT and other hydrocarbons such as alkanes, fatty acids, fats and oils

Dimorphic Fungi
• dimorphism- two forms of growth
• either as mold or yeast
• Mold-like (vegetative and aerial hyphae)
• Yeast-like (reproduce by budding)
• temperature dependent (37°C, yeast-like; 25°C, mold-like), Carbon dioxide dependent.

          

Mircobiology Week 8 and experiments

        Assalamualaikum and Good afternoon everyone.... Hw r u guys? I hope you all fine. 😊😊😊This is my eighth week of my microbiology class and my basic techniques of microbiology class. In Basic technique of microbiology class, we did experiment 11 which is differential staining for visualization of bacterial cell structures. On wednesday, we all had our hand's on test which is practical test about streak plate isolation and Gram staining. In microbiology, Dr Wan asked us to make some questions about our this week microbiology topic which is eukaryotic cells by group and other every groups must answer the questions. On friday, Dr Wan asked us to chose one organelle and related organelles with algae, protozoa and fungi. Then, Dr Wan asked us to send the link of journals that we found.😉😉😉

Basic techniques of microbiology
  This week for a change Dr Fairolniza is our lecturer until the our last week of basic techniques of microbiology class. My first impression about Dr Fairol is strict. But is actually opposite to my impression, Dr Fairol is so friendly, nice and also beautiful like Dr Adelene. Dr Fairol give some briefing and explanation about the experiment 11. We did our experiment 11 by our ownself without teaching by Encik Hussain and Encik Zainuddin. On wednesday, as usual we observe the slide we are smear and stain. And also we did our hand's on test. Hand's on test like the demo gave us one unknown bacterial cultural and we have to isolate by using streak plate method and staining using Gram staining method. After that, we have to observe the slide under microscope and write the observation in the paper that provided to us. Lastly, after we finished observing our experiments, our demo gave some examples how to write the references for lab reports.

EXPERIMENT 11: Differential staining for visualization of bacterial cell structures

Part A: Spore stain

      Certain bacterial species, most commonly gram-positive bacilli such as those of the genera Bacillus and Clostridium, undergo a complex developmental cycle that produces a resting endospore when faced with environmental adversity. The process of sporulation allows the bacteria to survive in harsh environmental conditions such as low nutrients, high temperatures, UV radiation, acids and toxic chemicals. If conditions improve, the spore may germinate to form a new vegetative cell and growth will resume. These cells have the capacity to undergo sporogenesis and give rise to a new intracellular structure called the endospore, which is surrounded by imprevious layers called spore coats. Endospores are very dehydrated structures that are not metabolically active. They possess a protein coat, called an exosporium, that forms a barrier around the spore. Since endospores are not easily destroyed by heat or chemicals, they define the conditions necessary to sterility. For example, to destroy endospores by heating, they must be exposed for 15-20 minutes to steam under pressure, which generates temperatures of 121° C. As conditions continue to worsen, the endospore is released from the degenerating vegetative cell and becomes an independent cell called a free spore. With the return of favourable environmental conditions, the free spore may revert to a metabolically active and less resistant vegetative cell through germination. 

Primary stain
Malachite green used as the primary stain. Malachite green unlike most vegetative cell types that stain by common procedures, the free spores, because of its impervious coats, will not accept the primary stain easily. For further penetration, the application of heat is required. After the primary stain is applied and the smear is heated, both the vegetative cell and spore will appear green.

Decolorizing Agent
Water is used as the decolorizing agent. Once the spore accepts the malachite green, it cannot be decolorized by tap water, which removes only the excess primary stain The spore remains green. On the outer hand, the stain does not demostrate a strong affinity for vegetative cell components; the water removes it, and these cells will be colorless.

Counterstain 
Safranin is used as the counterstain. This contrasting red stain is used as the second reagent to color the decolorized vegetative cells, which will absorb the counterstain and appear red. The spores retain the green of the primary stain. 

Part B: Capsule Stain

          For capsule staining, the capsule stain employs an acidic stain and a basic stain to detect capsule production. A capsule is a gelatinous outer layer that is secreted by the cell and that surrounds and adheres to the cell wall. Capsules are formed by organisms such as Klebsiella pneumoniae . Most capsules are composed of polysaccharides, but some are composed of polypeptides. The capsule differs from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. Some capsules have well-defined boundaries, and some have fuzzy, trailing edges. Capsules protect bacteria from the phagocytic action of leukocytes and allow pathogens to invade the body. If a pathogen loses its ability to form capsules, it can become avirulent. Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to stain the background using an acidic stain and to stain the cell itself using a basic stain. We use India ink and Gram crystal violet. This leaves the capsule as a clear halo surrounding a purple cell in a field of black. Capsule staining is more difficult than other types of differential staining procedures because the capsular materials are water-soluble and may be dislodged and removed with vigorous washing. Smears should not be heated because the resultant cell shrinkage may create a clear zone around the organism that is an artifact that can be mistaken for the capsule.

Primary stain

        Crystal violet (1% aqueous) is used as primary stain. A violet stain is applied to a non-heat fixed smear. At this point, the cell and capsular material will take on the dark color. 

Decolorizing agent

      Copper sulfate (20%) is used as decolorizing agent because the capsule is nonionic, unlike the bacterial cell, the primary stain adheres to the capsules but does not bind to it. The copper sulfate washes the purple primary stain out of the capsular material without removing the stain bound to the cell wall. At the same time, the decolorized capsule will now appear blue in contrast to the deep purple color of the cell. 

Capsule staining of Enterobacter aerogenes using nigrosin 

Capsule staining of Klebsiella pneumoniae using nigrosin 

Spore staining of Bacillus cereus 

Capsule staining of Klebsiella pneumoniae using crystal violet

Capsule staining of Enterobacter aerogenes using crystal violet

Microbiology week 8 ( Eukaryotic cell) 

Endoplasmic reticulum

New findings challenge current view on origins of Parkinson's disease

    It was found that the bulk of the damage to neurons with damaged mitochondria stems from a related but different source -- the neighbouring maze-like endoplasmic reticulum (ER). The ER has the important job of folding proteins so that they can do the vast majority of work within cells. Misfolded proteins are recognized by the cell as being dangerous. Cells halt protein production if there are too many of these harmful proteins present. While this system is protective, it also stalls the manufacture of vital proteins, and this eventually results in the death of neurons. To find out if ER stress might be at play in Parkinson's, a team led by Dr Miguel Martins analyzed fruit flies with mutant forms of the pink1 or parkin genes. Mutant forms of pink1 and parkin are already known to starve neurons from energy by preventing the disposal of defective mitochondria. These genes are also mutated in humans and result in hereditary versions of the disease. Much like Parkinson's patients, flies with either mutation move more slowly and have weakened muscles. The insects struggle to fly and they lose dopaminergic neurons in their brains -- a classic feature of Parkinson's. Compared to normal flies, Miguel's team found that the mutants experienced large amounts of ER stress. The mutant flies did not manufacture proteins as quickly as the non-mutants. They also had elevated levels of the protein-folding molecule BiP, a telltale sign of stress.


Lysosomes

Macrophage Lysosome Damage Crucially Contributes to Fungal Virulence

     Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans–induced lysosome damage was observed in infected murine bone marrow–derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans–containing lung cells compared with C. neoformans–free cells. Among C. neoformans–containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-g abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.

Peroxisome

Antifungal activity of Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA thiolase

       Peroxisomes play an important role in cellular defense systems and generate secondary messengers for cellular communication. Saccharomyces cerevisiae containing oleate-induced peroxisomes were subjected to buffer-soluble extraction and two chromatographicprocedures, and a protein with antifungal activity was isolated. The results of MALDI-TOF analysis identified the isolated protein as peroxisomal 3-ketoacyl-CoA thiolase (ScFox3). Purified yeast ScFox3 exhibited thiolase activity that catalyzed the thiolytic cleavage of 3-ketoacyl-CoA to acetyl-CoA and acylCoA. ScFox3 protein inhibited various pathogenic fungal strains, with the exception of Aspergillus flavus. Using ScFox3-GFP and PTS2 signal-truncated ScFox3M-GFP, we showed that only ScFox3-GFP, with an intact PTS2 peroxisome signal sequence, was able to translocate into peroxisomes. Yeast ScFox3 is a natural antifungal agent found in peroxisomes.

Vacuoles

Light Shielding in Blue-Green Algae

       Gas vacuoles are small, cylindrical, gas-filled vesicles which occur in certain procaryotic cells. They are found in numerous blue-green algae, many photosynthetic bacteria, some halophilic bacteria, and some planktonic freshwater bacteria. Because gas-vacuolate blue-green algae are often observed floating at the surface of water where high light intensity may damage cells, Lemmerman first suggested that gas vacuoles may function as light-shielding organelles. Recently, it has been suggested that such light shielding (if it occurred) might be due to the optical properties and the intracellular distribution of the gas vacuoles. Being gas filled, these vacuoles have a refractive index much lower than that of the cytoplasm which surrounds them, and therefore they scatter light. A suspension of gas vacuolate cells becomes visibly less milky and more transparent when the gas vacuoles are collapsed. Similarly, when a milky-white, opalescent suspension of isolated gas vacuoles is subjected to sudden pressure, a completely transparent suspension results. These changes are due to a decrease in light scattering when gas vacuoles collapse. But, although light scattering by gas vacuoles could protect cells by scattering away a large portion of the incident radiation, it could also lead to increased rather than decreased irradiation of some cellular components if the light were back-scattered into these components. Whether gas vacuoles function as light shields would depend upon the distribution of the gas vacuoles inside the cells.

Chloroplast

Toward mosquito control with a green alga: Expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas.

        We are developing Chlamydomonas strains that can be used for safe and sustainable control of mosquitoes, because they produce proteins from Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast. Chlamydomonas has a number of advantages for this approach, including genetic controls that are not generally available with industrial algae. The Bti toxin has been used for mosquito control for > 30 years and does not engender resistance; it contains three Cry proteins, Cry4Aa (135 kDa), Cry4Ba (128 kDa) and Cry11Aa (72 kDa), and Cyt1Aa (25 kDa). To express the Cry proteins in the chloroplast, the three genes were resynthesized and cry4Aa was truncated to the first 700 amino acids (cry4Aa700 ); also, since they can be toxic to host cells, the inducible Cyc6:Nac2-psbD expression system was used. Western blots of total protein from the chloroplast transformants showed accumulation of the intact polypeptides, and the relative expression level was Cry11Aa > Cry4Aa700 > Cry4Ba. Quantitative western blots with purified Cry11Aa as a standard showed that Cry11Aa accumulated to 0.35% of total cell protein. Live cell bioassays in dH20 demonstrated toxicity of the cry4Aa700 and cry11Aa transformants to larvae of Aedes aegypti and Culex quinquefasciatus. These results demonstrate that the Cry proteins that are most toxic to Aedes and Culex mosquitoes, Cry4Aa and Cry11Aa, can be successfully expressed in the chloroplast of Chlamydomonas.

Mitochondria

Hydrogen Production. Green Algae as a Source of Energy

       Hydrogen gas is thought to be the ideal fuel for a world in which air pollution has been alleviated, global warming has been arrested, and the environment has been protected in an economically sustainable manner. Hydrogen and electricity could team to provide attractive options in transportation and power generation. Interconversion between these two forms of energy suggests on-site utilization of hydrogen to generate electricity, with the electrical power grid serving in energy transportation, distribution utilization, and hydrogen regeneration as needed. A challenging problem in establishing H2 as a source of energy for the future is the renewable and environmentally friendly generation of large quantities of H2 gas. Thus, processes that are presently conceptual in nature, or at a developmental stage in the laboratory, need to be encouraged, tested for feasibility, and otherwise applied toward commercialization.

Nuclues

Regulation of eukaryotic DNA replication and nuclear structure

     In eukaryote, nuclear structure is a key component for the functions of eukaryotic cells. More and  more evidences show that the nuclear structure plays important role in regulating DNA replication. The nuclear structure provides a physical barrier for the replication licensing, participates in the decision where DNA replication initiates, and organizes replication proteins as replication factory for DNA replication. Through these works, new concepts on the regulation of DNA replication have emerged, which will be discussed in this minireview. Regulatory mechanisms for DNA replication are central to the control of the cell-cycle in eukaryotic cells. Recently, considerable progress has been made in our understanding of the relationship between regulation of eukaryotic DNA replication and nuclear structure. This review will briefly outline the progress and discuss some new concepts

appearing from the studies.