Bacterial Colony Formation, Metabolism and Enumeration

Bacterial Colony Formation  

Bacterial cells are microscopic organisms and as such are incapable of being seen with the naked eyes. As with all organisms, these organisms require nutrients to survive and reproduce. These nutrients can be found from various sources in the environment making these organisms ubiquitous. 
However, for experimental purposes, several culture media have been developed and incorporated with essential nutrients required for the growth of these organisms. These media may be semi-solid or liquid. While growing in the former, a single microscopic bacteria has the capacity to replicate itself in hundreds, thousands and eventually millions of cells which are bundled together. This is referred to as a colony and its appearance is governed by the characteristics of bacterium which serves as the building block of the colony. 
As such, the colony characteristics, generally referred to as colony morphology, serve as invaluable tools when trying to identify a bacteria species. Some colonial morphologies that are usually considered includes: shape, colour, edge/margin, texture, appearance, among others.

Metabolism in Microbial System 

Factors such as presence of nutrients, adequate temperature and pH are essential for the growth and reproduction of bacterial cell systems. These components interact with the an intricate network of enzymes and organelles in order to ensure continual survival of the bacterial cell, otherwise known referred to as metabolism. Metabolism  has two components namely,  catabolism  and  anabolism. 

1. Catabolism

Catabolism encompasses processes that harvest energy released from the breakdown of compounds (eg, glucose), and using that energy to synthesize  ATP. Catabolic processes include fermentation, photosynthesis and respiration.

a. Fermentation

Fermentation is characterized by substrate phosphorylation, an enzymatic process in which a pyrophosphate bond is donated directly to ADP (adenosine diphosphate) by a phosphorylated metabolic intermediate. The phosphorylated intermediates are formed by metabolic rearrangement of a fermentable substrate such as glucose, lactose, or arginine.
The Embden-Meyerhof (1), Entner-Doudoroff (2), and heterolactate (3) pathways are three pathways used for glucose catabolism in bacteria. In the absence of respiration or photosynthesis, bacteria are entirely dependent on substrate phosphorylation (fermentation) for their energy.

b. Respiration

Respiration requires a closed membrane. In bacteria, the membrane is the cell membrane. Respiration is energetically unfavorable and must be driven by a transmembrane electrochemical gradient, the proton motive force. Electrons are passed from a chemical reductant to a chemical oxidant through a specific set of electron carriers within the membrane, and as a result, the proton motive force is established; return of protons across the membrane is coupled to the synthesis of ATP. The biologic reductant for respiration frequently is NADH, and the oxidant often is oxygen. Compounds and ions other than oxygen may be used as terminal oxidants in anaerobic respiration. Suitable electron acceptors include nitrate, sulfate, and carbon dioxide

2. Anabolism

In contrast, anabolism or  biosynthesis , includes processes that utilize the energy stored in ATP to synthesize and assemble the subunits, or building blocks, of macromolecules that make up the cell such as cell envelope, peptidoglycan, capsules, among others. The sequence of building blocks within a macromolecule is determined in one of two ways. 

  1. In nucleic acids and proteins, it is  template-directed : DNA serves as the template for its own synthesis and for the synthesis of the various types of RNA; messenger RNA serves as the template for the synthesis of proteins. 
  2. In carbohydrates and lipids, on the other hand, the arrangement of building blocks is determined entirely by enzyme specificities. Once the macromolecules have been synthesized, they self-assemble to form the supramolecular structures of the cell, eg, ribosomes, membranes, cell wall, flagella, and pili.   

Regulation of Metabolic Pathways

The rate of macromolecular synthesis and the activity of metabolic pathways must be regulated so that biosynthesis is balanced. All of the components must be controlled in order to ensure that the resources of the cell are not expended on products that do not contribute to growth or survival. Regulation of metabolic processes can be achieved by:

  1. Feedback Inhibition: The end-product in each case allosterically inhibits the activity of the first—and only the first—enzyme in the pathway.
  2. Allosteric Activation: In some cases, it is advantageous to the cell for an end product or an intermediate to activate rather than inhibit a particular enzyme. In the breakdown of glucose by E coli, for example, overproduction of the intermediates G6PD and phosphoenolpyruvate signals the diversion of some glucose to the pathway of glycogen synthesis; this is accomplished by the allosteric activation of the enzyme converting glucose 1-phosphate to ADP-glucose.
  3. Enzyme Inactivation: This can be achieved by covalent modification of some enzymes through processes such as adenylation and phosphorylation.

Enumeration of Bacteria

This refers to the different methods through which the number of bacteria in a sample can be quantified. Since each colony arises from a single bacteria cell, the number of colonies counted per sample is recorded in colony forming units (CFU). Bacteria cell counting includes total cell counting and viable cell count.

Total Bacteria Cell Count

These methods are used for counting both dead and live bacteria. They include

  1. Electronic Counters: These includes the Coulter Counter. They are used for counting samples with high microbial bioburden. Here, the broth culture is forced through an orifice in the chamber of the counter. The chamber is designed such that electric current passes through it and passage of the individual cells disrupts the current with each disruption being recorded and measured.
  2. Turbidometric Method: This is done by measuring the turbidity of the broth culture, using a spectrophotometer (measured in Optical Density Units).

Viable Bacteria Cell Count

These methods are used to determine the number of cells that survive in a medium. 
The Plate Count method is used to determine the number of viable micro-organisms in a sample. Here, the samples are inoculated on the agar plate and the number colonies formed are counted after incubation. The dilution factor and amount of sample inoculated on the medium should also be taken note of and applied to the following formula:
Number of microorganisms = number of colonies formed X total dilution factor
The result is written in colony forming units/volume of inoculum.
The viable bacteria cell count methods includes:

  1. Membrane Filter Method: A membrane filter contains pores which traps bacteria. The sample is filtered through the membrane filter, after which the latter is placed on an agar plate. The number of colonies are counted after incubation.

This method is less time-consuming and can be used to examine large sample volumes. 

  1. Bacterial Spore Count Method: Here, the sample is preheated to kill off the vegetative cells. The sample is then placed on an agar plate, incubated and number colonies counted.
  2. Pour Plate Method: This method involves the mixing of a set volume of the sample with molten agar. The agar is left to set, incubate and colonies counted. In order help ensure the presence of distinct colonies, it may be necessary to dilute the samples before inoculating.
  3. Surface Spread Plate Method: The sample is spread over the surface of the agar, incubated and the number of colonies counted. 

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