Basic techniques of microorganisms
Many methods have been devised to accomplish this, including direct microscopic counts, use of an electronic cell counter such as the Coulter Counter, chemical methods for estimating cell mass or cellular constituents, turbidimetric measurements for increase in cell mass, and the serial dilution-agar plate method.Direct microscopic counts are possible using special slides known as counting chambers. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (>107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity. A variation of the direct microscopic count has been used to observe and measure growth of bacteria in natural environments. In order to detect and prove that thermophilic bacteria were growing in boiling hot springs, T.D. Brock immersed microscope slides in the springs and withdrew them periodically for microscopic observation. The bacteria in the boiling water attached to the glass slides naturally and grew as microcolonies on the surface. Direct microscopic counts require the use of a specialized slide called the Petroff-Hausser counting chamber, in which an aliquot of a eukaryotic cell suspension is counted and the total number of cells is determined mathematically. Breed smears are used mainly to quantitate bacterial cells in milk.
Electronic cell counter is an example of an instrument capable of rapidly counting the number of cells suspended in a conducting fluid that passes through a minute orifice through which an electric current is flowing. Cells, which are nonconductors, increase the electrical resistance of the conducting fluid, and the resistance is electronically recorded, enumerating the number of organisms flowing through the orifice. In addition to its inability to distinguish between living and dead cells, the apparatus is also unable to differentiate inert particulate matter from cellular material.
Chemical methods is while not considered means of direct quantitate analysis, chemical methods may be used to indirectly measure increases both in protein concentration and in DNA production. In addition cell mass can be estimated by dry weight determination of a specific aliquot of the culture. Measurement of certain metabolic parameters may also be used to quantitate bacterial populations.
Spectrophotometric analysis is increased turbidity in a culture is another index of growth. With turbidimetric instruments, the amount of transmitted light decrease as the cell population increases, and the decrease in radiant energy is converted to electrical energy and indicated on galvanometer. This method is rapid but limited because sensitivity is restricted to microbial suspensions of 10 million cells or greater.
Serial dilution-agar plate analysis is while all these methods may be used to enumerate the number of cells in a bacterial culture. To accomplish this, the serial dilution-agar plate technique is used briefly this method involves serial dilution of a bacterial suspension in a sterile water blanks, which serve as a diluent of known volume. Molten agar, cooled to 45℃, is poured into a Petri dish containing a specified amount of the diluted sample. Dilutions should be plated in dilutions for greater accuracy, incubated overnight, and counted on a Quebec colony counter either by hand or by an electronically modified version of this instrument. Plates suitable for counting must contain not fewer than 30 nor more than 300 colonies. The total count of the suspension is obtained by multiplying the number of cells per plate by the dilution factor, which is the reciprocal of the dilution. Advantages of the serial dilution-agar plate technique are only viable cells are counted and it allows isolation of discrete colonies that can be subcultured into pure cultures, which may then be easily studied and identified. Disadvantages of this method are overnight incubation is necessary before colonies develop on the agar develop on the agar surface, more glassware is used in this procedure and the need for greater manipulation may results in erroneous counts due to errors in dilution or plating.
Microbiology (viruses)
A viral species is a group of viruses sharing the same genetic information and ecological niche
• Family: Rhabdoviridae, Genus: Lysavirus, Species: rabies virus
• Family: Retroviridae, Genus: Lentivirus, Species: human immunodeficiency virus
Characteristics to divide viruses into taxonomic groups
- Nature of host
- Nucleic acid characteristics
Virus Family
DNA viruses
• Adenoviruses, Herpesviruses Papovaviruses and Hepadnaviruses.
• DNA of most DNA viruses is released into the nucleus of the host cell.
• Transcription and translation produce viral DNA and later capsid protein which is synthesizes in the cytoplasm.
RNA viruses
• Multiplication occurs in the cytoplasm
• RNA-dependent RNA polymerases synthesizes the ds RNA.
• Picornaviruses, Togaviruses, Rhabdoviruses and Retroviruses.
• After maturation, viruses are released by budding or through ruptures in the host cell membrane.
Acellular microorganims
Characteristics: What are viruses ?
• Latin for “poison”
• smaller than bacteria
– NOT retained by bacterial filters (filterable)
– NOT visible in the light microscope
• obligately intracellular parasites
– NOT cultivable in vitro on any nutrient medium
– increase and multiply only in living tissue/cells
Distinctive Feature of Viruses
• contain a single type of nucleic acid, either DNA/RNA but never both
• contain a protein coat (sometimes itself enclosedby an envelope of lipids, proteins and carbohydrates) that surrounds the nucleic acid
• multiply inside living cells by using the synthesizing machinery of the host cell
• cause the synthesis of specialized structures that can transfer the viral nucleic acid to other cells
Virions and viroids
• virion:
– intact, fully assembled, infective virus
• viroid:
– piece of RNA without a protein coat responsible for several plant diseases
Host Range
• Can infect invertebrates, vertebrates, plants, protists, fungi OR bacteria
• possess narrow host range
• determined by presence of specific receptors on the cell and availability of host cellular factors for viral multiplication
• some are very specific
Size
• smaller than bacteria (almost all)
• wide range
• Picornaviridae
– 27 nm (foot and mouth disease virus)
• Poxviridae
Viral structures
- Nucleic acids
- Capsid
- Envelope
Capsid and Envelope
• Capsid: protein coat surrounding the nucleic acid
• composed of subunits, capsomers, which can be a single type of protein or several types
• Envelope covered the capsid: consisting of lipids, proteins and carbohydrates
• Spikes: structures that protrude out of the envelope
General Morphology (capsid architecture)
• Helical viruses
• Polyhedral viruses
• Enveloped viruses
• Complex viruses
Replication/multiplication of bacteriophages
• Lytic cycle
– host cells lyse and die
• Lysogenic cycle
– host cell remains alive
Viroids
- Plant infected.
- Viroid – an infectious RNA particle smaller than a virus.
- Viroids have been found to differ from viruses in six ways:
- Viroids consists of a single circular RNA molecule of low molecular weight.
- Viroids exist inside cells as particles of RNA without capsids or envelopes.
- Viroids do not require a helper virus.
- Viroid RNA does not produce proteins.
- Viroid RNA is always copied in the host cell nucleus.
- Viroid particles are not apparent in infected tissues without the use of special techniques to identify nucleotide sequences in the RNA
Prions (proteinaceous infectious particle)
• Prions believed to consist of a single type of protein with no nucleic acid component. The prion protein and the gene which encodes it are also found in normal 'uninfected' cells.
• These agents are associated with infectious and inherited diseases, such as Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy (BSE) in cattle (mad cow disease)
Characteristics of prions
• Resistant to inactivation by heating to 90℃, which will inactivate virus.
• Prion infection is not sensitive to radiation treatment that damages virus genomes.
• Prions are sensitive to protein denaturing agents, such as phenol and urea
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