Microbiology Week 14

Assalamualaikum and Good morning.... How r you everyone? I hope everyone will be fine as well. 😊😊😊 This is fourteenth week of my microbiology class. In microbiology, we study about the nutritional types of microorganisms.

2 major nutritional concern:
a.Source of energy
b.Source of carbon

Energy sources
• phototrophs: light as primary energy source
• chemotrophs: inorganic and organic compunds as primary energy source (from redox reactions)

Carbon sources
• Autotrophs (self feeders): CO2 – lithotrophs
• heterotrophs (feeders on others): organic carbon – organotrophs

Combine the energy and carbon sources:
• Photoautotrophs
• Photoheterotrophs
• Chemoautotrophs
• Chemoheterotrophs

Photolithoautotrophy
- Photolithotrophic autotrophs or photoautotrophs
- Source of energy: Light Source of C: CO2 Source of e-: reduced inorganic molecules e.g. Algae and cyanobacteria: H2O Purple and green sulfur bacteria: H2, H2S and elemental S

Photoorganoheterotrophy
- Photoorganotrophic heterotrophs
- Source of energy: Light Source of C: Organic C molecules Source of e-: Organic molecules
- Common inhabitants of polluted lakes/streams

Chemolithoautotrophy
- Chemolithotrophic autotrophs
- Source of energy: Oxidation of organic/inorganic compounds Source of C: CO2 Source of e-: Reduced inorganic molecules (iron, N, S)
- Contribute to chemical transformations of elements in the ecosystem

Chemoorganoheterotrophy
- Chemoorganotrophic heterotrophs or chemoheterotrophs
- Source of energy: Oxidation of organic/inorganic compounds Source of C: Organic C molecules Source of e-: Organic molecules
- Same organic nutrient à satisfy all requirements
- Most pathogenic m/os

Mixotrophic
- Alter metabolic patterns in response to environmental changes
- E.g. Purple nonsulfur bacteria Photoorganoheterotroph
    – absence of O2 Chemoorganoheterotrophs
    – presence of O2 The term mixotroph can describe organisms (usually algae or bacteria) capable           of  deriving metabolic energy both from photosynthesis and from external energy sources. These         organisms may use light as an energy source, or may take up organic or inorganic compounds.

Photoautotroph
• Photosynthetic bacteria, algae, cyanobacteria and green plants.
• obtain energy from photophosphorylation and fix carbon from CO2 via the Calvin-Benson cycle to synthesize organic compounds.
• photosynthetic bacteria:
– oxygenic phototrophs, produces O2 : cyanobacteria.
– anoxygenic phototrophs, do not produce O2, anaerobic condition: green sulfur bacteria (eg. Chlorobium) and purple sulfur bacteria (Chromatium)
• Green sulfur bacteria
– Chlorobium
– Use sulfur (S), sulfur compounds (H2S) or hydrogen gas (H2) to reduce CO2 and form organic compounds.
• Purple sulfur bacteria
– Chromatium
– Also use sulfur, sulfur compounds or H2 to reduce CO2.
– They are distinguished from the green sulfur bacteria by the location of their bacteriochlorophyls, location of stored sulfur and ribosomal RNA

Photoheterotrophs
• use light as an energy source and an organic compound for their carbon source or electron donor
• They are anoxygenic
• purple nonsulfur bacteria (Chloroflexus)
• green non sulfur bacteria (Rhodopseudomonas)

Chemoautotrophs
• use inorganic compounds as their energy source and CO2 as their carbon source
• Inorganic sources of energy:
– H2S : Beggiatoa
– S : Thiobacillus thioxidans
– NH3 : Nitrosomonas
– NO2 - : Nitrobacter
– H2 : Hydrogenomonas
– Fe2+ : Thiobacillus ferrooxidans
• energy stored in ATP - produced by oxidative phosphorylation

Chemoheterotrophs
• use complex organic molecules as their carbon and energy sources
• The energy source and carbon source are usually the same organic compound (glucose)
• most bacteria and all fungi, protozoa and animals
– saprophytes: live on dead organic matter
– parasites: derive nutrients from living host

          

Microbiology Week 13

Assalamualaikum and Good morning.... How r you everyone? I hope everyone will be fine as well. 😊😊😊 This is thirteenth week of my microbiology class. In microbiology, we study about the microbial growth.

Growth vs. Tolerance
 – Growth - referring to the number of cells, not the size.
 – Tolerance - survive under conditions in which they cannot grow.
 – The suffix “-phile” describe conditions permitting growth, whereas the term “tolerant” describes conditions in which the organisms survive, but don’t necessarily grow.
 – E.g. a “thermophilic bacterium”, “thermotolerant bacterium”

The requirements for growth
 • Physical requirements
 - light, temperature, pH, water activity and osmotic pressure.
 • Chemical requirements
 - electron donor (C, N, S, P, K)
 - electron acceptor (O2 - , NO3- , SO4 2-, CO3 - , Fe3+) - micronutrients (vitamins, amino acids, trace minerals)

Trace elements - e.g. Fe, Mg, Mn, Cu, Zn …
- Microorganism dependant
- Important for enzyme function
- part of enzyme and cofactors
- catalysis of reactions
- maintenance of protein structure
- Organic growth factors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines

Special growth factors
- Specifically needed for growth of certain m/os
- e.g. Legionella pneumophila (Legionaires’ disease)
 ~ absolute requirement for a.a. L-cystein and iron
 ~ in nature
 ~ provided by algae and amoebas

Aseptic technique
 Definition Aseptic technique refers to carrying out a procedure under controlled conditions in a manner that will minimize the chance of contamination. Contaminants may be introduced from the environment, equipment and supplies, or personnel.

 Aseptic Technique
 • Sterile Hood - All manipulations must be carried out in a sterile cabinet – Turn the UV light off (Should ordinarily be on) – Open the cabinet – Wipe down with disinfectant (70% ethanol or 40% isopropyl alcohol)
 • Bring materials into the hood
 • Light up the flame or gas
 • Begin your work
 • Flame all caps and lids
 • Tightly close all boples and caps
 • Remove materials from the hood
 • Turn off gas
 • Wash the hood surface
 • Turn the UV light on to disinfect

Culturing Microorganisms
 • There are two basic culture techniques used in microbiology:
 1. Liquid culture: bacteria, algae, and some fungi can be reared in culture tubes (test tubes) in a liquid medium.
~ Liquid medium is best when you want to rapidly increase the concentration of the organism or when you want to grow motile cells.
 2. Culture Plates: Liquid medium is solidified using agar (agarose) and poured as a thin layer in the bopom of a culture dish (also some=mes called petri plate)
 ~ Culture plates are used when you want to test (1) antibiotic sensitivity, (2) estimate culture concentrations from environmental samples, or (3) isolate individual colonies from environmental samples

Culturing bacteria
 • Culturing bacteria in the laboratory present two problems:
 –To obtain pure culture
 –To use suitable medium

Pure culture
 • Is a population of identical cells originating from a single cell.
 • Pure cultures are obtained by working in aseptic environments.

Anaerobic Environments
 • Reducing Compounds
 – Thioglycholate
 – Cystein
 – Anything with – SH
 – Must Use Indicator
 • Gas Pack (anaerobic generating kit)

Anaerobic Incubation
 • Anaerobic Jar
 – Impermeable to Oxygen
 • Catalyst
 – Platinum or Palladium
 – In Lid or on Gas Pack
 • Gas Pack
 – Uses Oxygen and Replaces with Carbon Dioxide

New technique to provide anaerobic environment
 • Oxyrase
 – Reduces oxygen to water
 – Respiratory enzyme derived from the plasma membranes of certain bacteria
 – Added into growth medium
 • Avoid the need for more cumbersome apparatus

Culture media
 CULTURE MEDIUM
- A nutrient material prepared for the growth of microorganisms in a laboratory.
 INOCULUM
 - Microbes that are introduced into a culture medium to initiate growth.
 CULTURE
- The microbes that grow and multiply in or on a culture medium.
 STERILE
- No living microbes

Types of Culture Media
 • Chemically defined vs. complex media
 – Chemically defined media
 • The exact chemical composition is known • e.g. minimal media used in bacterial genetics experiments
 – Complex media
 • Exact chemical composition is not known
 • Oten consist of plant or animal extracts, such as soybean meal, milk protein, etc.
 • Include most routine laboratory media, e.g., tryptic soy broth.
 • Liquid (broth) vs. semisolid media
 – Liquid medium
 • Components are dissolved in water and sterilized
 – Semisolid medium
 • A medium to which has been added a gelling agent
 • Agar (most commonly used)
 • Gelatin
 • Silica gel (used when a non-organic gelling agent is required)
 • Selective media
 – Contain agents that inhibit the growth of certain bacteria while permiting the growth of others
 – used to isolate specific organisms
 • Differential media
 – Contain indicators that react differently with different organisms (for example, producing colonies with different colors)
 – Used in identifying specific organisms
 • Enrichment media
– The enrichment media will increase the small numbers of desired bacteria to detectable level.
– One type of bacteria present in small numbers while the other type present in much larger numbers. – The enrichment medium is usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe but not others.

      

Microbiology Week 12 and Experiments

       Assalamualaikum and Good morning.... How r you everyone? I hope everyone will be fine as well. 😊😊😊 This is twelveth week of my microbiology class and and my basic technique of microbiology class. In basic technique of microbiology class, we did our last experiment for 1st semester is experiment 18 which is Serial Dilution-Agar Plate Procedure to Quantitate Viable Cells. In microbiology we study about the virus and acellular microorganisms and study about sterilization and disinfectants.

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