Spore stain

 

Some Gram-positive bacteria produce endospores which are highly resistant to heating and a variety of chemicals. These spores are found in the genera Bacillus and Clostridium. The spores are difficult to stain using normal techniques, but it is possible to drive the stain into them using heat. Once the stain has been taken up by the spores, it is extremely difficult to decolourise them, although the vegetative portion of the cell can be decolourised easily. Several versions of the spore stain are used.

Gram Stain

This is the most important stain in bacteriology and is so central to identification that it should be practised until the operator is fully competent. A number of different variations are found, and the laboratory should standardise on one method.

A General Model for Biofilm Development

Biofilm formation is a developmental process in which bacteria undergo a regulated lifestyle switch from a nomadic unicellular state to a sedentary multicellular state where subsequent growth results in structured communities and cellular differentiation. Results of prior work by many groups allow the construction of a hypothetical developmental model for biofilm formation that can be generalized for many different bacterial species. This model can be adjusted to fit either of two general modes of unicellular lifestyle: nonmotile and motile.

Transport of an Infectious Agent

Transmission involves the transport of an infectious agent from the reservoir to the host. It is the most important link in the chain of infection. Pathogens can be transmitted from the reservoir to a susceptible host by various routes (Sobsey and Olson, 1983).

a. Person-to-Person Transmission

The most common route of transmission of infectious agents is from person to person. The best examples of direct contact transmission are the sexually transmitted diseases such as syphilis, gonorrhea, herpes, or acquired immunodeficiency syndrome (AIDS).

Determination of Cell Biochemicals

Microbial biomass can also be measured by determination of specific cell biochemical constituents such as ATP, DNA, RNA, proteins, phospholipids, bacterial cell wall components, or photosynthetic pigments (Sutton, 2002).

a.        ATP

Adenosine triphosphate has often been used to determine live microbial biomass in environmental samples, using a ratio of C/ATP = 250 for aquatic samples. However, the ATP content of cells varies with the growth rate and metabolic state of microorganisms and nutrient limitation. A better measure is the total adenylate pool AT (AT = ATP + ADP + AMP) because it does not change greatly with changes in metabolic activities of the microorganisms. The adenylate energy charge (EC) ratio provides information on growth potential of naturally occurring microbial populations.

Fungi (Eukaryote)

Fungi are eukaryotic organisms that produce long filaments called hyphae, which form a mass called mycellium. Chitin is a characteristic component of the cell wall of hyphae. In most fungi, the hyphae are septate and contain crosswalls that divide the filament into separate cells containing one nucleus each. In some others,the hyphae are nonseptate and contain several nuclei. They are called coenocytic hyphae.

Unusual Types of Bacteria (Part 3)

. Actinomycetes

Actinomycetes are gram-positive filamentous bacteria characterized by mycelial growth (i.e., branching filaments), which is analogous to fungal growth. However, the diameter of the filaments is similar in size to bacteria (approximately 1 mm). Most actinomycetes are strict aerobes, but a few of them require anaerobic conditions. Most of these microorganisms produce spores, and their taxonomy is based on these reproductive structures (e.g., single spores in Micromonospora or chains of spores in Streptomyces). They are commonly found in water, wastewater treatment plants, and soils (with preference for neutral and alkaline soils). Some of them (e.g., Streptomyces) produce a

Unusual Types of Bacteria (Part 2)

. Gliding Bacteria

These filamentous gram-negative bacteria move by gliding, a slow motion on a solid surface. They resemble certain cyanobacteria except that they are colorless. Beggiatoa and Thiothrix are gliding bacteria that oxidize H2S to S0, which accumulates as sulfur granules inside the cells. Thiothrix filaments are characterized by their ability to form rosettes. Myxobacteria are another group of gliding microorganisms. They feed by lysing bacterial, fungal, or algal cells. Vegetative cells aggregate to make “fruiting bodies,” which lead to the formation of resting structures called myxospores. Under favorable conditions, myxospores germinate into vegetative cells.

. Bdellovibrio (B. bacteriovorus)

These small (0.2–0.3 mm), flagellated (polar flagellum) bacteria are predatory on gram-negative bacteria. After attaching to the bacterial prey, Bdellovibrio penetrates the cells and multiplies in the periplasmic space (space between the cell wall and the plasma membrane). Because they lyse their prey, they are able to form plaques on a lawn of the host bacterium. Some Bdellovibrio can grow independently on complex organic media.

Unusual Types of Bacteria (Part 1)

. Sheathed Bacteria

These bacteria are filamentous microorganisms surrounded by a tubelike structure called a sheath. The bacterial cells inside the sheath are gram-negative rods that become flagellated (swarmer cells) when they leave the sheath. The swarmer cells produce a new sheath at a relatively rapid rate. They are often found in polluted streams and in wastewater treatment plants. This group includes three genera: Sphaerotilus, Leptothrix, and Crenothrix. These bacteria have the ability to oxidize reduced

DNA Replication and Protein Synthesis

Replication: The DNA moleculecan make an exact copy of itself. The two strands separate and new complementary strands are formed. The double helix unwinds and each of the DNA strands acts as a template for a new complementary strand. Nucleotides move into the replication fork and align themselves against the complementary bases on the template. The addition of

Cytoplasmic Membrane (Plasma Membrane)

The cytoplasmic membrane is a 40–80 A ˚ -thick semipermeable membrane that contains a phospholipid bilayer with proteins embedded within the bilayer (fluid mosaic model) (Fig. 1.3). The phospholipid bilayer is made of hydrophobic fatty acids oriented towards the inside of the bilayer and hydrophilic glycerol moieties oriented towards the outside of the bilayer. Cations such as

HACCP

There is growing acceptance throughout the EU and in many other countries of the value of HACCP principles in ensuring the microbiological safety of foods. The HACCP approach is a systematic way of analysing the potential hazards of a food operation, identifying the points in the operation where the hazards may occur, and where controls over those that are important to consumer safety can be achieved. Most of the product-specific EC directives as well as the Directive on the Hygiene of Foodstuffs (93/43/EEC), place obligations on industry and food business operators to adopt HACCP principles as the basis for their product safety management systems. The advantages of the HACCP approach over a food safety control system based purely on microbiological standards is now widely recognized. Thus, the Commission proposes to consolidate and simplify existing EC food hygiene legislation. These are expected to be implemented by 2004. The proposed consolidation adopts a unified approach to hygiene and extends the general hygiene rules and HACCP principles to cover hygiene throughout the food chain, including primary production, i.e. the ‘farm-to-fork’ approach to managing food safety. Responsibility of food safety will be unambiguously placed onto food producers. A fully documented HACCP plan will be required of all food producers, including caterers, regardless of size.

This will include a specific monitoring programme, thereby reinforcing the own-check principle of food producers. An absolute requirement for full traceability of all foods and ingredients used in food production is also introduced, such that all food producers must keep adequate records to allow full traceability throughout the products’ allotted shelf-life.

Measurement of Active Cells in Environmental Samples

Several approaches have been considered for assessing microbial viability/activity in environmental samples. Epifluorescence microscopy, in combination with the use of oxido-reduction dyes, is used to determine the percent of active cells in aquatic environments. The most popular oxido-reduction dyes are INT (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (cyanoditolyl tetrazolium chloride) (Poschet al., 1997; Pyle et al., 1995a). A good correlation was found between

Measurement of the Number of Viable Microbes on Solid Growth Media

This approach consists of measuring the number of viable cells capable of forming colonies on a suitable growth medium. Plate count is determined by using the pour plate method (0.1–1 mL of microbial suspension is mixed with molten agar medium in a petri dish), or the spread plate method (0.1 mL of bacterial suspension is spread on the surface of an agar plate). The results of plate counts are expressed as colony forming units (CFU). The number of CFU per plate should be between 30 and 300. Membrane filters can also be used to determine microbial numbers in dilute samples. The sample is filtered and the filter is placed directly on a suitable growth medium.

                                     

Culture-based methods have been routinely used in soil, aquatic, and wastewater microbiology, but they reveal only about 0.1–10 percent of the total bacterial counts in most environments (Pickup, 1991). Indeed, some microorganisms (e.g., E. coli, Salmonella typhimurium, Vibrio spp.) can enter into the viable but nonculturable (VBNC) state and are not detected by plate counts, especially when using selective growth media (Koch, 2002; Roszak and Colwell, 1987). The VBNC state can be triggered by factors such as nutrient deprivation or exposure to toxic chemicals. This phenomenon is particularly important for pathogens that may remain viable in the VBNC state for longer periods of time than previously thought. The VBNC pathogens may remain virulent and cause disease in humans and animals.

Total Number of Microbial Cells

Total number of cells (live and dead cells) can be measured by using special counting chambers such as the Petroff–Hauser chamber for bacterial counts or the Sedgewick–Rafter chamber for algal counts. The use of a phase-contrast microscope is required when nonphotosynthetic microorganisms are under consideration. Presently, the most popular method consists of retaining the cells on a membrane filter treated to suppress autofluorescence (use of polycarbonate filters treated with Irgalan Black) and staining the cells with fluorochromes such as acridine orange (AO) or 40,6-diamidino-2-phenylindol (DAPI). The microorganisms are subsequently counted using an epifluorescence microscope (Kepner and Pratt, 1994).

An advantage of DAPI is its stable fluorescence. A wide range of other fluorochromes are available for many applications in environmental microbiology studies. These include, among others, PicoGreen, SYBR-Green 1 and 2, Hoechst 33342, YOYO-1, and SYTO dyes (green, red, and blue) (Neu and Lawrence, 2002).

Scanning electron microscopy (SEM) has also been considered for measuring total microbial numbers. Electronic particle counters are also used for determining the total number of microorganisms in a sample. These instruments do not differentiate, however, between live and dead microorganisms, and very small cells may be missed. Flow cytometers are fluorescence-activated cell sorters and include a light source (argon laser or a mercury lamp) and a photodetector, which measures fluorescence (use correct excitation wavelength) and scattering of the cells. They sort and collect cells with predefined optical parameters. They are often used in the biomedical and aquatic microbiology fields (Paul, 1993). They have been used to sort algal cells and to distinguish between cyanobacteria from other algae, based on phycoerythrin (orange) and chlorophyll (red) fluorescence. They can help identify microorganisms when combined with fluorescent antibodies.