Cultivation methods - Microbial Cultivation

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Chapter: Pharmaceutical Microbiology : Fundamental features of microbiology

Most bacteria and some yeasts divide by a process of binary fission whereby the cell enlarges or elongates, then forms a cross wall (septum) that separates the cell into two more or less equal compartments each containing a copy of the genetic material.


CULTIVATION METHODS

 

Most bacteria and some yeasts divide by a process of binary fission whereby the cell enlarges or elongates, then forms a cross wall (septum) that separates the cell into two more or less equal compartments each containing a copy of the genetic material. Septum formation is often followed by constriction such that the connection between the two cell compartments is progressively reduced (see Figure 2.1a) until finally it is broken and the daughter cells separate. In bacteria this pattern of division may take place every 25–30 minutes under optimal conditions of laboratory cultivation, although growth at infection sites in the body is normally much slower owing to the effects of the immune system and scarcity of essential nutrients, particularly iron. Growth continues until one or more nutrients is exhausted, or toxic metabolites (often organic acids) accumulate and inhibit enzyme systems. Starting from a single cell many bacteria can achieve concentrations of the order of 109 cells ml−1 or more following overnight incubation in common liquid media. At concentrations below about 10 7 cells ml−1 culture media are clear, but the liquid becomes progressively more cloudy (turbid) as the concentration increases above this value; turbidity is, therefore, an indirect means of monitoring culture growth. Some bacteria produce chains of cells, and some produce elongated cells (filaments) that may exhibit branching to create a tangled mass resembling a mould mycelium (Figure 2.1d). Many yeasts divide by budding (see section 1.2.3 and Figure 2.1b) but they, too, would normally grow in liquid media to produce a turbid culture. Moulds, however, grow by extension and branching of hyphae to produce a mycelium (Figure 2.1c) or, in agitated liquid cultures, pellet growth may arise.

 

When growing on solid media in Petri dishes (often referred to as ‘plates’) individual bacterial cells can give rise to colonies following overnight incubation under optimal conditions. A colony is simply a collection of cells arising by multiplication of a single original cell or a small cluster of them (called a colony forming unit or CFU). The term ‘colony’ does not, strictly speaking, imply any particular number of cells, but it is usually taken to mean a number sufficiently large to be visible by eye. Thus, macroscopic bacterial colonies usually comprise hundreds of thousands, millions or tens of millions of cells in an area on a Petri dish that is typically 1–10 mm in diameter (Figure 2.1 e). Colony size is limited by nutrient availability and/or waste product accumulation in just the same way as cell concentration in liquid media. Colonies vary between bacterial species, and their shapes, sizes, opacities, surface markings and pigmentation may all be characteristic of the species in question, so these properties may be an aid in identification procedures .

 

Anaerobic organisms may be grown on Petri dishes provided that they are incubated in an anaerobic jar. Such jars are usually made of rigid plastic with airtight lids, and Petri dishes are placed in them together with a low-temperature catalyst. The catalyst, consisting of palladium coated pellets or wire, causes the oxygen inside the jar to be combined with hydrogen that is generated by the addition of water to sodium borohydride; this is usually contained in a foil sachet that is also placed in the jar; alternatively, oxygen may be removed by combination with ascorbic acid. After its removal, an anaerobic atmosphere is achieved and this is monitored by an oxidation– reduction (redox) indicator; resazurin is frequently used as a solution soaking a fabric strip.

 

Yeast colonies often look similar to those of bacteria, although they may be larger and more frequently coloured. The appearance of moulds growing on solid microbiological media is similar to their appearance when growing on common foods. The mould colony consists of a mycelium that may be loosely or densely entangled depending on the species, often with the central area (the oldest, most mature region of the colony) showing pigmentation associated with spore production (Figure 2.1f). The periphery of the colony is that part which is actively growing and it is usually nonpigmented.

 


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