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Techniques Manual Table of Contents

Physiological Processes of Bacteria

Introduction

The Cell Walls of Gram Positive and Gram Negative Bacteria. Bacteria are unique among all organisms in the composition of their cell walls. Composed of a material called peptidoglycan, the bacterial cell wall is essentially a tremendously long polysaccharide which wraps around the bacterial cell membrane. The parallel strands of polysaccharide are cross linked by short peptides to provide additional structural strength. The peptidoglycan may be present in a single layer (Gram negative bacteria)or in multiple layers (Gram positive bacteria), sometimes as many as 12 layers. Gram positive and Gram negative bacteria also have many fundamental differences in their physiology and ecology. Furthermore, the medical treatment of Gram positive and Gram negative bacterial infections is also frequently different. For example, many of our antibiotics target cell wall formation and drugs which work against Gram positive cell wall formation are generally ineffective against Gram negatives. For these reasons, whenever bacteria are being characterized, we start by finding their Gram's stain characteristics and cellular morphology.

Bacteria are an excellent model system for studying physiological processes. Most metabolic pathways proceed in the same fashion in all organisms. Because bacteria are easy to work with and grow so rapidly they are ideal for many types of metabolic studies. In addition, discerning which metabolic pathways a particular species of bacteria uses is an important differential tool for identification purposes. In this exercise we will look at several important physiological processes such as whether the bacteria use oxidative respiration or fermentation (or both), the production of particular waste products and the utilization of particular metabolites.

One key physiological determination to be made when working with bacteria is to assess their requirement for oxygen. Some bacteria absolutely need oxygen to live (aerobes), others are killed by a faint whiff of oxygen (obligate anaerobes) while others can grow with oxygen but can also grow (albeit more slowly) in its absence (facultative anaerobes). There are also aerotolerant anaerobic bacteria like the streps which can live in the presence of oxygen but don't use it.

Anaerobic bacteria (either obligate or facultative) produce ATP without the assistance of Krebs Cycle or the oxidative electron transport system. Many will therefore get all their ATP from fermentation, meaning they produce lots of acidic waste products. We can therefore differentiate between aerobes and facultative bacteria based on detecting these acidic waste products. Obligate anaerobes can't grow without special media to remove all the oxygen so we will not be working with any of these. Beside, anaerobic bacteria really stink!

The SIM media is extremely useful for looking at several physiological characteristics. SIM is an acronym for sulfur, indole and motility. This media allows us to simultaneously determine whether these two metabolic waste products are being produced and whether the bacteria is motile. Sulfur and indole are produced when amino acids are used for energy sources. The amino acids methionine and cysteine contain sulfur. When they are used for energy, the bacteria excrete excess sulfur in the form of hydrogen sulfide (H2S). H2S reacts with iron in the SIM agar and forms a black precipitate. Indole (part of the amino acid side chain) is produced as a waste product when bacteria use the amino acid tryptophan.


Experimental

Part One. Gram Stain Characteristics.

Once we have isolated colonies of bacteria, the next step need to characterize the bacteria by using the Gram stain. Practice the gram stain technique by staining the known gram positive and gram negative bacteria provided by your instructor. Once you are comfortable with the technique, Gram stain your unknown pure culture isolate. and determine its Gram stain characteristics and verify its cellular morphology.

Part Two: Oxygen Utilization.

On a PER TABLE basis, inoculate one known aerobe and one known facultative anaerobe to phenol red broth tubes. These are your control tubes. In addition, each student will inoculate his/her unknown to a separate phenol red broth. Record your results after 24 hour incubation

Part Three: The SIM media.

Each table of students should inoculate SIMs with the two control bacteria provided by the instuctor and each of the unknowns. After 24 to 48 hours, check for the following characteristics:

  • Motility. Motile bacteria will make the whole media turbid while non-motile bacteria will grow only along the stab line.
  • Sulfur. Excretion of sulfur will turn the media black.
  • Indole. Excretion of indole is detected by adding Kovac's Reagent to the top of the media AFTER the incubation period. The presence of indole will turn the Kovac's Reagent bright red.

Part four. Exoenzyme production. Some bacteria produce exoenzmes to help them degrade macromolecules from the environment so that the simple nutrients can be transported across the cell membrane. One example of an exoenzyme is amylase, the enzyme responsible for breaking down starch into smaller units, primarily glucose and maltose, the glucose disaccharide. Each student should inoculate their unknown bacteria along with a known starch positive and negative bacteria onto a starch agar and obtain results after 24 - 48 hours.