Objectives for Today's Class		Mastery Topics
Reference: Agrios Chapter 12		Plant Diseases Caused by Bacteria Examples

1. General Characteristics of Bacteria

   1. What is a Prokaryote?

      Generally, a single-celled microorganism that has a cell membrane, or membrane and a cell wall surrounding it but has no organized nucleus (not membrane enclosed) or subcellular organellar structures.

   2. General Structure

      1. Glycocalyx
         1. Composed of Polysaccharides: Most are glucose polymers (dextrans) or fructose polymers (levans)
         2. Functions: Protects cell against dehydration; Protects against phagocytosis: increases virulence
      2. Cell Wall
         1. Present in all prokaryotes - except mycoplasmas
         2. Function:
         • Gives shape;
         • Coccus
         • Bacillus
         • Spiral
         • Protects against osmotic lysis
         • Serves as a primer for new cell wall synthesis
         • Provides a receptor site for viral attachment
         • Some cell wall components are endotoxins
         3. Structure: The rigidity is due to the presence of a substance called Peptidoglycan (composed of amino-sugars and amino acids).
         
         
         4. Cell Wall Types: Gram + and Gram -
         
         
         • Gram Stain - Differential Stain
         The difference in reaction is due to the differences in the cell wall structure of Gram + and Gram - Cells. The Gram + cell has a single thick wall layer, through which the decolorizing solvent (alcohol) does not readily penetrate. The alcohol dehydrates the peptidoglycan resulting in a decrease in the porosity of the cell wall. The results in retention of the crystal violet-iodine complex. The Gram - cell wall has a wall with several thinner layers through which the colorizer readily penetrates.

         Results: Gram +: retains crystal violet and appears purple
         .................Gram -: Decolorizes and retains the counterstain, safranine, thus appearing red/pink.

      3. Plasmalemma (Inner Membrane): This structure is composed primarily of Lipid and protein. The membrane is fluid in nature allowing the composite molecules to move within, through, and across it. It separates the inside from the outside of the cell and functions both as a permeability barrier and transport facilitator.

      4. Nucleoid (a region): Prokaryotes do not possess a true nucleus. There is NO nuclear envelope. The DNA molecule is in a high coiled condition and is called a chromosome. If the DNA of a single bacterial cell could be uncoiled and displayed linearly it would be about 2mm long, which is about 1000 times the length of the bacterium. Thus it is apparent that the DNA is high coiled (supercoiled) and very highly organized in order for it to function within the cell.

         Other smaller DNA molecules, including plamids and episomal DNA, provide extra-chromosomal "genetic" components to the cell. They are not necessarily intimately associated with the Nucleoid.

      5. Ribosomes: Small particles composed of protein and highly organized with RNA. Part of the translation apparatus, and synthesis of all proteins take place on these structure. Ribosomes in prokaryotes are 70S. This is an arbitrary unit based on their sedimentation characteristics in an ultracentrifuge. "S" refers to Svedberg Unit. This term is valuable for determining density properties of particles but should not be taken necessarily as indicative of relative size between two particles. The 70S particle is made of two smaller subunits; one 30S and the other 50S. Eukaryotic ribosomes are 80S and made up of 40S and 60S subunits.

         The structural compositional differences between 70S (prokaryote) and 80S (eukaryote) ribosomes forms an important basis or using antibiotics.

         For an excellent presentation on tRNA and Ribosomes; click HERE

      6. Fimbriae (Pili): THin, smaller and more numerous that flagella. Protein in nature. Functions in attachment, sexual conjugation, and as specific sites for viral recognition and attachment.

      7. Flagella: Are made up of a single protein (flagellin). Long and thin appendages and elongate from the tip.

         Position of the flagella is used as an aid in identification and classification of bacteria.

      8. General Functions of Bacteria

         1. Media

            : a substance containing all the nutrients required for the growth of a microorganism
            1. Types of media based on composition
               • Simple (defined): all components are known
               • Complex (natural): contains undefined components, i.e. yeast extract, casein (milk proteins)
            2. Types of media based on consistency
               • Liquid - Broth cultures, Natural Fluids
               • Solid - usually a semi-solid provided by agar ( a polysaccharide extracted from algae)
                 • Reasons agar is the support medium of choice
                   • No nutrient value,
                   • Is not degraded during culture
                   • Melts at ~100C and solidifies at ~44C ( with the advent of Molecular Biology, agars have been very highly developed such that both the melting and solidification temperatures have been modified for specific applications.
                   • Medium can be incubated over a wide range of temperatures
                   • Transparent
            3. Types based on Function
               • Selective Media: favors growth of a particular type of organism while either not supporting or inhibiting the growth of others.

                 In essence ALL media are Selective media, simply because not all organisms will grow on all media.

               • Differential medium: Allows one to "differentiate" among organisms based on their growth
               • Enrichment medium: Favors growth of a particular type of organism without the use of inhibitors of others.
               • General purpose: allows for growth of a variety of organisms.

         2. Staining

            1. Reasons for staining
               • Makes the organisms easier to visualize
               • Enable differentiation between organisms; i.e. Gram stain
               • Enable differentiation of structures
            2. General Stains
               1. Positive Stain, Simple Stain, or Single Stain
                  • Purpose of fixation:
                    • Causes organisms to adhere to slide
                    • Kills organisms
                    • May modify cell structures to allow for better dye uptake
                  • Drawbacks to fixation:
                    • May modify cell structures
                    • Shrinks cells
               2. Negative Stain: The background is stained and the microorganisms appear light against a dark background. This can be quite useful for visualizing extra cellular appendages that will be damages with other procedures.

                  BOTH Positive and Negative stains depend on the fact that bacteria have net negative charge associated with their outer surfaces. largely because of the phosphate groups of the cell membrane.

               3. Differential Stains
                  • Gram Stain:
                    • Most Phytopathogenic bacteria are gram negative; i.e. Pseudomonas, Xanthomonas, Erwinia,
                    • Most coccid are Gram positive
                    • Gram positive cultures tend to become Gram negative with age; therefore, use only fresh young cultures for Gram stains.

                  • Acid Fast Staining:
                    • The key component necessary is a unique lipid fraction called Mycolic acid. Most acid fast organisms are found in the genus Mycobacterium.

                  • Stains for specific cell structures
                    • There are many stains for specific structures; i.e. cell wall, flagella, capsule, etc
            3. Spores are often "stained for" using Malachite Green

         3. Methods of Isolation Axenic Cultures

            "All media is Selective"

            We have considered types of media. Before one uses it to isolate microorganisms, one must consider how to exclude unwanted (or contaminant) organisms. Microbes are everywhere. Because of their small sizes they are easily dispersed in the air, carried on one's person (skin, clothes, hair, etc), infuse liquids, and abound on surfaces.

            1. Sterilize the medium: The most reliable, and most common, method is by heat (autoclave - heat under pressure; 15 min and 121C). Sterilization can also be obtained by filtration through a 0.2 millimicron filter.
            2. Aseptic Transfer Technique: By aseptic technique, microbiologists mean taking all prudent precautions to prevent contamination of the culture as well as cross contamination of oneself and equipment.
               • Clean the work area: Disinfectant (remember disinfectants would be of no value if they didn't kill cells; therefore, because you are made of cells be careful not to get it on yourself.
               • Sterilize transfer instruments: Flame sterilization is effective and most common. It is not necessary to melt the instrument of affect sterilization.
               • Work quickly and efficiently
               • Work quietly, this is not the time to "Whistle while you work"

         4. Several Different Methods for the Isolation of Pure Cultures

            1. Streak Plate: The key to this method is that one creates a dilution gradient with successive streaks. Begriming with an initial streak of the desired culture, one streaks single across the plate; removes and flame sterilizes the transfer needle, turns the culture plate 30 degrees and streaks through the preceding streak; one continues the flaming and streaking for a total of five streaks.

            2. Spread Plate: One places a drop of a microbial suspension in the center of an agar medium plate and uses a glass rod to uniformly distribute the drop over the surface of the plate. The key here is to use plates that have desiccate slightly on their surface so that discrete colonies will form.

            3. Pour Plate : Microbial suspension is added to a melted agar tube (careful, if the agar is too hot one will only kill the microbes; but if too cold the agar will solidify - practice and experience). The suspension and agar are mixed well and poured in to sterile petri dishes using aseptic techniques. The agar solidifies, trapping the organisms within the matrix of the medium. While the agar is firm enough to hold the organism, it is porous enough to permit growth of colonies.

         5. Maintaining Pure Cultures

            1. The microorganisms can be subcultures periodically onto or into fresh medium to permit continued growth. This is time consuming and because organisms are infamous for mutation in culture it is not the best long term method.

            2. Short-term storage (up to 30 days) - Refrigerate

            3. Long-term storage - Freeze in liquid nitrogen; Lyophilization (freeze-drying)

      9. Antimicrobial Agents

         1. Antiseptics and Disinfectants

            Antiseptics can be used on one's skin, Disinfectants are restricted to inanimate objects

            1. Halogens - chlorine; oxidized cell components - iodine; binds and inactivates proteins

            2. Alcohols - disrupt the plasmalemma; dehydrate the cell; precipitate proteins

            3. Heavy metals - Combine with and inactivate proteins

               
               

            4. Phenolic compounds - inactivate proteins
            5. Cationic detergents, Quaternary Ammonium Compounds - disrupt the plasmalemma

         2. Antibiotics

            One considers toxicity, spectrum of action (broad vs narrow), dosage, and cost.
            1. Cell Wall Targeting Antibiotics - inhibitors of peptidoglycan synthesis (inhibits formation of the peptide cross-linkages)
               • Penicillin
               • Cephalosporin
            2. Cell Membrane Targeting Antibiotics - the plasmlemma is the site of action
               • Polymyxin - changes the structure of the plasmalemma causing leakage
            3. Protein Synthesis Inhibitors - Specifically act on 70S ribosomes
               • Streptomycin - binds to 30S ribosomal subunit causing misreading of the RNA
               • Tetracycline
               • Chloramphenicol - binds to 50S subunit preventing peptide bond formation
            4. Nucleic Acid Synthesis Targeting Antibiotics
               • Naladixic acid - interferes with DNA replication at the replication fork

         3. Methods by which Antibiotic Resistance Occurs

            1. The organism lacks the structure on which the antibiotic acts. Mycoplasmas have no cell walls; therefore, penicillin is useless for their control.
            2. The organism is impermeable to the antibiotic
            3. The antibiotic is inactivated. Penicillin resistance when an organism produces an enzyme called beta-Lactamase (or penicillinase) with breaks down the penicillin.
            4. The target in modified so the antibiotic can't bind. Streptomycin resistance occurs when there is a change in the 30S subunit which is responsible for binding the antibiotic.

Exposition page on Naming bacteria

Questions, Comments, Complaints and Complements?

Drafted by Lisa Wolfson Keith , Teaching Assistant 1995.

This page is authored and maintained by:

Dr. J.E. Partridge, Department of Plant Pathology, University of Nebraska-Lincoln

jpartridge1@unl.edu Home Page References