Metabolic Diversity Two fundamental nutritional needs: Acquiring chemical energy in the form of ATP Pump ions, move cargo, undergo chemical reactions in the cell Obtaining carbon Used to synthesize fatty acids, DNA, RNA, and other molecules Bacteria are the masters of metabolism. As a group they can eat almost anything, from hydrogen molecules to crude oil. ATP = adenosine triphosphate Millions of species obtain the energy required to make ATP by oxidizing organic compounds like sugars, starch, or fatty acids. Really it comes down to the movement of electrons. As a highly reduced compound (such as sugar) accepts more and more electrons, it builds up a potential energy gradient. This energy is used to produce ATP. In essence this is called respiration. Carbon is used to synthesize fatty acids, DNA, RNA, and other materials needed to build cells. There are a number of different pathways to obtain energy and carbon - 6 , in fact – including photoautotrophs, chemolithotrophs, and chemoorganotrophs. In other words, a number of bacteria use compounds other than sugars to create energy. For instance, hydrogen sulfide and methane.

Culturing Techniques Researchers use specific compounds and substrates in the culture medium and observe what grows! MacConkey agar Isolates enteric, Gram-negative, bacteria based on their ability to ferment lactose The composition of the cell wall varies among bacteria and is an important character for identifying and classifying bacteria. Peptidoglycan is a layer of carbohydrates cross-linked by proteins. Bacteria with a fairly thick wall made of this material retain a purple color when stained with a dye known as crystal violet, and are known as Gram-positive. Gram-negative bacteria have double cell walls, with a thin inner wall of peptidoglycan and an outer wall of carbohydrates, proteins and lipids. They do not stain purple with crystal violet. MacConkey agar = bile salts and crystal violet inhibit the growth of Gram-positive organisms. Organisms that ferment lactose create an acidic environment, creating pink/red colonies (neutral red is a pH indicator also included in the MacConkey agar). Non-fermenters will produce normally colorless colonies.

SIM Test S = Sulfur reduction I = Indole production M = Motility Kovac’s reagent M = Motility SIM medium is a combo of three different parameters It contain nutrients, iron and sodium thiosulfate. If an organism can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron to form ferric sulfide, which is a black precipitate. If black is present, it is a positive result for sulfur-reducing bacteria. One of the nutrients in the SIM medium is peptone, which contains amino acids including tryptophan. Some bacteria can break down tryptophan to produce indole. Kovac’s reagent reacts with indole to produce a red compound. Motility is the ability to move. If you see diffuse growth outward from the stab line, you have a positive for motility.

Simmons Citrate Test Tests the ability of certain organisms to utilize citrate as a carbon source. pH indicator = Bromthymol blue pH > 7.5 turns royal blue Neutral pH green (just like uninoculated media)

Christensen Urea Test Some bacteria utilize urea, which results in the formation of ammonia. Alkaline Produces a red-pink/red-violet color A negative reaction will have no color change and the agar medium will remain pale yellow.

Identifying Specific Bacteria Bacteria Genus Sulfur Indole Lactose Citrate Urease Escherichia - + Edwardsiella +/- Salmonella Enterobacter

Water Quality Index Fecal coliform measurements: Multiple-Tube Fermentation Most Probable Number (MPN) Membrane Filtration Measures both Total Coliform and E. coli

Membrane Filtration using mColiblue