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Friday, March 10, 2017

Alagae, Fungi, Parasite

Entamoeba histolytica 

Entamoeba histolytica is an anaerobic  parasitic  amoebozoa, part of the genus Entamoeba.  Predominantly infecting humans and other primates causing amoebiasis. Inside humans Entamoeba histolytica lives and multiplies as a trophozoite. Trophozoites are oblong and about 15–20 µm in length. In order to infect other humans they encyst and exit the body. The life cycle of Entamoeba histolytica does not require any intermediate host. Mature cysts (spherical, 12–15 µm in diameter) are passed in the feces of an infected human. Another human can get infected by ingesting them in fecally contaminated water, food or hands. 








Aspergillus sp.,
Aspergillus is a genus consisting of a few hundred mold species found in various climates worldwide. spergillum is an asexual spore-forming structure common to all Aspergillus species; around one-third of species are also known to have a sexual stage. Aspergillus is defined as a group of conidial fungi—that is, fungi in an asexual state

Human and animal disease
Aspergillus causes  lung disease, called Aspergillosis, Aspergillosis is, in particular, frequent among horticultural workers who inhale peat dust, which can be rich in Aspergillus spores. It has been found in the mummies of ancient Egyptian tombs and can be inhaled when they are disturbed

Industrial uses

Aspergillus  is cultured for the industrial production of many substances. Various strains of A. niger are used in the industrial preparation of citric acid (E330) and gluconic acid (E574) and have been assessed as acceptable for daily intake by the World Health Organisation. A. niger fermentation is "generally recognized as safe" (GRAS) by the United States Food and Drug Administration under the Federal Food, Drug, and Cosmetic Act.
 Many useful enzymes are produced using industrial fermentation of A. niger. For example, A. niger glucoamylase is used in the production of high-fructose corn syrup, and pectinases are used in cider and wine clarification.
 Some Aspergillus species are
A.niger,  A.flavus , Aspergillus fumigates, A. terreus etc.,



  





Penicillium Sp.,

Penicillium is a genus of ascomycetous fungi of major importance in the natural environment as well as food and drug production. Some members of the genus produce penicillin, a molecule that is used as an antibiotic, which kills or stops the growth of certain kinds of bacteria inside the body. Other species are used in cheesemaking.
The thallus (mycelium) typically consists of a highly branched network of multinucleate, septate, usually colorless hyphae. Many-branched conidiophores sprout on the mycelia, bearing individually constricted conidiospores. The conidiospores are the main dispersal route of the fungi, and often are green in color.
Economic Value : 
Several species of the genus Penicillium play a central role in the production of cheese and of various meat products. To be specific, Penicillium  molds are found in Blue cheese. Penicillium camemberti  and  Penicillium roqueforti  are the molds on Camembert, Brie, Roquefort, and many other cheeses.  Penicillium nalgiovense  is used to improve the taste of sausages and hams, and to prevent colonization by other molds and bacteria.[17]
In addition to their importance in the food industry, species of Penicillium and Aspergillus serve in the production of a number of biotechnologically produced enzymes and other macromolecules, such as gluconic, citric, and tartaric acids, as well as several pectinases, lipase, amylases, cellulases, and proteases. Some Penicillium species have shown potential for use in bioremediation because of their ability to break down a variety of xenobiotic compounds.
The genus includes a wide variety of species molds that are the source molds of major antibiotics. Penicillin, a drug produced by P. chrysogenum (formerly P. notatum), was accidentally discovered by Alexander Fleming in 1929, and found to inhibit the growth of Gram-positive bacteria (see beta-lactams).





  

Rhizopus Sp., 


Rhizopus is a genus of common saprophytic fungi on plants and specialized parasites on animals. They are found on a wide variety of organic substrates, including "mature fruits and vegetables",[2] jellies, syrups, leather, bread, peanuts, and tobacco. 
Rhizopus species grow as filamentous, branching hyphae that generally lack cross-walls (i.e., they are coenocytic). They reproduce by forming asexual and sexual spores. In asexual reproduction, sporangiospores are produced inside a spherical structure, the sporangium. Sporangia are supported by a large apophysate columella atop a long stalk, the sporangiophore. Sporangiophores arise among distinctive, root-like rhizoids. In sexual reproduction, a dark zygospore is produced at the point where two compatible mycelia fuse. Upon germination, a zygospore produces colonies that are genetically different from either parent.
·         R. microsporus var. oligosporus is used to make tempeh, a fermented food derived from soybeans.
·         R. oryzae is used in the production of alcoholic beverages in parts of Asia and Africa.
·         Rhizopus stolonifer (black bread mold) causes fruit rot on strawberry, tomato, and sweet potato and used in commercial production of fumaric acid and cortisone.




Chlamydomonas 
Chlamydomonas is a genus of green algae consisting of unicellular flagellates, found in stagnant water and on damp soil, in freshwater, seawater. It is generally found in habitat rich in ammonium salt. Chlamydomonas possesses red eye spots for photosensitivity and reproduces by both asexual and sexual means.
Chlamydomonas's asexual reproduction goes through Zoospores, Palmella stage, Aplanospores and Hypnospores; sexual reproduction through isogamy, anisogamy or oogamy.

Nutrition

Most species are obligate phototrophs but C. reinhardtii and C. dysosmos are facultative heterotrophs that can grow in the dark in the presence of acetate as a carbon source.

Morphology

·         Motile unicellular algae.
·         Generally oval in shape.
·         Cell wall is made up of glycoprotein and non cellulosic polysaccharides instead of cellulose.








Tuesday, February 14, 2017

ENDOSPORE STAINING

ENDOSPORE STAINING (Schaeffer-Fulton method)

Aim:  

To determine the spore forming (Endospore) bacteria by Schaeffer-Fulton method

Principle :

Endospore staining is used to visualize specialized cell structures. The endospore stain is used to determine the highly resistant spores of certain microorganisms within their vegetative cells. The multiple thick coats of the spore made the endospore resistant to stain with most dyes. In Schaeffer-Fulton method, the primary stain, Malachite Green, is added over the heat fixed bacterial smear and heated over a steam bath for few minutes. This will soften the hard outer coverings of the spore and the primary stain gets stick to the spore. When taken from the steam bath followed by further cooling hardens the outer layer of the spore. During this stage both the spore and vegetative cells appear as green in color. But later the thick outer layer makes the spore resistant to the action of decolorizing agent (water), but however, water can easily decolorize the vegetative cells.  When counterstained with Safranin, vegetative cells are easily stained with Safranin, and the cells appear in red or pink color.

Materials required
Clean glass slide in box
Inoculation loop
Test organism
Absorbent paper
Boiling water in water bath
Water in wash bottle
Microscope
Primary Stain – Malachite green
Counter stain - Safranin


Procedure

1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.

2. Air dry and heat-fix the bacterial smear.

3. Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.
Place the slide over a container of boiling water.

4. Saturate the absorbent paper with malachite green stain solution and steam for 5 minutes.  Keep the paper moist by adding more stain as required.

5. Remove the absorbent paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds.

6. Wash the slide with water.

7. Counter stain with safranin for 30 seconds.

8. Wash the slide with water and blot dry the slide.

9. Examine the slide under the oil immersion lens for the presence of endospores.

Result: 

 On microscopic observation endospores appear in green color and the vegetatives cells as pink.




Micrometry

           Determination of Microbial Cell Size by Micrometry
Aim:
            To measure the dimensions of microorganisms with the help of a microscope.
 Principle: 
Microorganisms are microscopic objects that are visible only with the help of a microscope.  Sometimes it is necessary to measure its dimensions (length breadth and diameter) for its identification process. But, determination of the size of a microorganism is not an easy process. Micrometry refers to the measurement of dimensions of the desired microorganisms under a microscope which uses two micro-scales known as ‘micrometers’. At first, the diameter of the microscopic field must be established with the help these micrometers namely ocular micrometer and stage micrometer. Ocular micrometer with microscopic graduations etched on their surfaces is a circular glass disc that fits into the circular shelf inside the eyepiece of the microscope. It has 100 equally spaced divisions marked as 0 to 10. Depending on the objective being used, the distance between these graduations will vary that determines the size of the field. The stage micrometer is clipped to the stage of the microscope.  In the centre of the stage micrometer a known 1mm distance is etched into100 equally spaced divisions making each division equals 0.01 mm or 10 µm. 
Calibration:
            The calibration procedure for the ocular micrometer to requires that the graduations on both micrometers to be superimposed on each other. The number of ocular divisions that corresponds to the known distance in the stage micrometer is determined. Finally the calibration calculated as follows.
                                                              Number of divisions on stage micrometer
One division of ocular micrometer = ____________________________________ x10
                                                               Number of divisions on ocular micrometer
After calibration, the ocular micrometer measures the size of various microbes including its length, breadth, and diameter. First count the number of spaces occupied by the organism on ocular micrometer graduations. Then multiply this number by calibration factor.

Materials Required:
Stage micrometer
Ocular micrometer
Microscope
Microscopic slide
Microbial culture

Procedure
  1. The eyepiece is removed from the microscope, and its top lid is unscrewed. The lid is removed. Carefully, the eye lens is removed. The ocular micrometer (a circular etched glass piece, which slips into the eye piece) is placed carefully into the eyepiece. The eye lens is placed back and the top lid is screwed to its original condition. The eyepiece is placed back in the microscope.
  2. The stage micrometer is clipped to the stage and the etchings centered by moving the mechanical stage.
  3. Adjust ocular and the objectives (10x, 45 x, 100x) which is to be calibrated.
  4.  Superimpose the graduation of the slide with that of the ocular such that the line on the slide at one end exactly coincides with first line on the ocular. This done by rotating ocular and moving the stage micrometer.
  5. Count the number ocular meter divisions coinciding between two lines of both ocular micrometer and stage micrometer.
  6. The size of the bacterial cell is determined by counting the number of divisions in ocular micrometer occupies by single cell.




Result:
The size of the given microbial cell determined as ___________ (length, breadth) in 10x/40x/100x.




Wednesday, January 11, 2017

BACTERIAL PIGMNETS ( CELL BIOLOGY)



Bacterial Pigments 

Bacteria are pigmented or colored. Pigmented bacteria are also known as chromobacteria. Bacterial pigments are water soluble or insoluble; water soluble pigments are diffused in the growth medium. Chemically, bacterial pigments are pyrrole, phenazine, carotenoid, xanthophylls and quinine or quinone derivatives. The pigment molecules are synthesized in cell wall or periplasmic space. We can visualize pigmentation in bacteria in specific growth medium or by staining bacterial cells with a dye to observe under microscope. It has been proved that only aerobic and facultatively aerobic bacteria are pigmented because, molecular oxygen is essential for pigmentation. Therefore, anaerobic bacteria are nonpigmented. Pigment synthesis is also dependent on light, pH, temperature and media constituents like indicator dyes.
Examples for Pigmented Bacteria:
 
Purple: Spirillum rubrum
Violet: Chromobacterium violacein
Blue: Streptomyces coelicolor (actinorhodin edible)
Green: Chlorobium tepidum
Yellow: Xanthomonas campestris (xanthomonadins)
Orange: Sarcina aurentiaca
Red: Serratia marcescens (prodigiosin)
Black: Prevotela melaninogenica
Golden: Staphylococcus aureus
Silver: Actinomyces sp.
White: Staphylococcus epidermidis
Cream: Proteus vulgaris
Pink: Micrococcus roseus
Fluorescent blue/green: Pseudomonas aeruginosa (Pyocyanin)
Fluorescent yellow: Pseudomonas fluorescens (Pyoverdin/fluorescein) 




Detection of bacterial pigments on indicator media: Various types of indicator or differential media are used during the culturing of bacteria. These media are made by using suitable pH indicator dyes such as methylene blue, eosin, methyl red and chemicals like sodium sulphite, potassium tellurite, which change color when bacteria are cultured in them. When bacteria are cultured on indicator medium, growth is observed as colored colonies. MacConkey agar, EMB agar, McLeod agar and TCBS agar are some of the examples of routine indicator media used in Microbiology. On MacConkey agar lactose fermenting bacteria show pink pigmented colonies while as lactose non-fermenters are colorless. These media are very useful in identification and biochemical characterization of particular bacteria.

Why do bacteria produce pigments?

Pigmentation is very useful for bacteria as well as for our industries. In bacteria, pigment formation is associated with morphological characteristics, cellular activities, pathogenesis, protection and survival. Autotrophic cyanobacteria contain a green colored pigment, known as chlorophyll (similar to plant chlorophyll). They also contain accessory pigments phycobilin and chlorophyll b which are required in photosynthesis. Other photosynthetic bacteria have pigments bacteriochlorophyll, proteorhodopsin and bacteriorhodopsin similar to chlorophyll. It seems that in autotrophic bacteria, pigments are needed to carry out the process of photosynthesis. Pigments of photosynthetic bacteria carry out photosynthesis similarly like plant chlorophyll.

Pigments are produced by bacteria to absorb UV radiation or to quench oxygen free radicals. In both the cases bacterial pigment play important role of the cell protection. Some bacterial pigments are antibiotics which are active against phytopathogenic fungi, bacteria, and yeasts; also active against human pathogenic Gram positive or negative bacteria and fungi. Pigments prodigiosin (Serratia), erythromycin (from Streptomyces), pyocyanin, pyoverdin and pyochelin from Pseudomonas, spirilloxanthin of Spirillum are potent antibiotic pigments.

Bacterial pigments help in survival in stress conditions. For example, in rhizosphere region iron is always present in limited amount; rhizobacteria like pseudomonads produce iron chelating compounds or siderophores. Siderophores scavenge traces of iron and make available to host plant. They also eradicate pathogenic fungi and bacteria by depriving them for iron. Pigments produced by Pseudomonas spp. like pyoverdin and pyochelin act like siderophore. Extremophiles are very colorful. Bright pigmentation of extremophilic bacteria offers protection from oxidative stress. Pigments also maintain membrane integrity and stability. The pigments of extremophiles are also required in respiratory or photosynthetic functions.

Pigments confer antibacterial and heavy metal resistance. Pathogenic staphylococci are multidrug resistant because of their pigment which acts as barrier for antibiotics acting on cell wall and plasma membrane. Bacteria showing heavy metal resistance are usually pigmented as they have been exploited for remediation of soil and water polluted by heavy metals like arsenic, copper, cadmium, mercury and nickel. Pigmented bacteria have also been used as biosensors to detect environmental pollution like oil spills or pesticide and heavy metal recalcitrance.
 

Many important applications of bacterial pigments are enlisted:

In pathogenesis:
Ø Resistance to phagocytosis
Ø Heat resistance and acid stability
Ø Unpalatability to protozoa
Ø In vitro antibody formation enhancers
Ø Antitumor properties

Industrial applications:
Ø In paint formulations
Ø Alternatives to color additives of plant origin
Ø In textile dyeing
Ø Food colorants
Ø Source of vitamin A
Ø In therapeutics
Ø Indicators of oil spill
Ø Biosensors and markers of water, soil and air pollution