BACTERIAL STRUCTURE, PHYSIOLOGY, AND GENETICS BACTERIAL CELL WALL: From the inside out.

GRAM-POSITIVE: Plasma Membrane: Contains transport enzymes. In oxidative bacteria, contains proteins for oxidative phosphorylation. Contains no sterols. Serves as the attachment site for segregation of DNA (cytokinesis). Peptidoglycan Cell Wall: MUREIN is the name of the cross-linked substance making up the cell wall. TEICHOIC ACID is also contained within the Peptidoglycan wall. PROTOPLAST forms when a bacterium is insensitive to lysis from Lysozyme which normally lyses the bacterial cell wall. Forms when the cell is in a medium isotonic with its interior. This is one form of environmentally-induced bacterial resistance. GRAM-NEGATIVE Plasma Membrane PERIPLASMIC SPACE: That area between plasma membrane and peptidoglycan sheath. Peptidoglycan Layer: Much thinner than the Gram-Positive Peptidoglycan wall. It has been greatly reduced, with part of it contributing to the periplasmic gel. LIPOPOLYSACCHARIDE (LPS) COAT: Outer covering of gramnegatives. Endotoxin: LPS coat is the basis for gram-negative bacterial endotoxin, which causes anaphylaxis. BAYER'S JUNCTIONS: Zones of adhesion between the outer and inner membrane. SPHEROPLASTS result in gram-negatives if you attempt to kill resistant cells with lysozyme. Part of the periplasmic space remains intact. ACID-FAST BACTERIA: Mycobacteria. Share similar staining properties as Gram-Positive. MYCOLIC ACID: They have an outer coat of waxy material made of Mycolic Acid, which is extremely impermeable. CELLULAR STRUCTURES: CAPSULE: Polysaccharide slime that can evade host defenses and adhere to surfaces. They are virulence factors. Streptococcus Pneumonia FLAGELLA: Helical protein made of flagellin Types of Flagella: Monotrichous: Single flagellum. Lophotrichous: Two or several flagella oriented in a bipolar, linear fashion. Peritrichous: Flagella oriented all about the cell. Basal Body: Intracellular structure that anchors the flagellum.
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PILI: Extensions of cytoplasmic membrane with little openings. Made of pilin. Common Pili SEX PILI: Special kind of pili. CYTOSOL: Ribosomes: 30S + 50S subunits = 70S ribosomes. SPORE: Produced by some Gram positive rods. It is produced in response to poor nutrition. It is made of a coat that is keratin-like and rich in Cys. Has special chemical called dipicolinic acid. BACTERIAL GROWTH: TEMPERATURE PSYCHROPHILES: Grow best at cold temperature. MESOPHILES: Grow best at medium temperature. All pathogenic bacteria are mesophiles. THERMOPHILES: Grow best at hot temperature. BACTERIAL SECRETIONS: Extracellular Proteases are secreted to degrade host ECM, lipid, DNA, and proteins. EXOTOXINS: Secreted substances that exhibit host toxicity. A-B TOXINS: Examples = Cholera Toxin, Diphtheria Toxin. Exotoxins that ultimately act intracellularly. STRUCTURE: Two major subunits. The B portion binds to the cell and the A portion is injected into cell to exhibit toxic effect. MEMBRANE-DISRUPTING TOXINS: Pore-forming toxins. No enzymatic activity -- only ability to insert into cell membrane. SUPERANTIGENS: Toxins of superantigen class that indiscriminately bind to MHC-II and TCR, and thus induce polyclonal activation of T-Cells. Examples: Staph Enterotoxin Strep Exotoxin-A TSS Toxin BACTERIAL CELL TRANSPORT: PASSIVE TRANSPORT ACTIVE TRANSPORT: Active means of transporting molecule (nutrients such as lactose) into the bacterial cell. IRON TRANSPORT: SIDEROPHORES, iron-sequestering proteins, are secreted by bacterial cells. BACTERIAL METABOLISM: Embden-Meyerhof Glycolysis: Standard pyruvate-forming anaerobic glycolysis Streptococcus Mixed-Acid Fermentation: Form formic acid and acetic acid as byproducts. Escherichia Coli Enterobacteriaceae Butanediol Fermentation: Enterobacter Butyric Acid Fermentation: Clostridium

Aerobes: Mycobacterium Tuberculosis OXYGEN NUTRITION: FREE RADICAL OXYGEN: SUPEROXIDE DISMUTASE breaks down the Superoxide Anion, O2-2. It is required for bacteria to survive in the presence of oxygen. CATALASE breaks down Hydrogen Peroxide, H2O2. It is not required, but some bacteria possess it. OBLIGATE ANAEROBES: Bacteria that undergo anaerobic fermentation and cannot survive in the presence of oxygen. They do not have Superoxide Dismutase, thus O2 is poisonous as it will yield oxidative radicals that cannot be broken down. OBLIGATE AEROBES: Bacteria that undergo strictly oxidative respiration and require oxygen for survival. They do have Superoxide Dismutase and thus can survive in O2 FACULTATIVE ANAEROBES: Bacteria that can undergo fermentation in the absence of O2, or respiration in its presence. They do have Superoxide Dismutase and thus can survive in O2. AEROTOLERANT ANAEROBES: Bacteria that never undergo oxidative respiration but can nonetheless tolerate the presence of oxygen. They do have Superoxide Dismutase. Streptococcus Pneumonia is an aerotolerant bacterium that has superoxide dismutase but not catalase. MICROAEROPHILIC: They are facultative anaerobes, but they prefer low O2concentration conditions. Flagellar Movement RUN: Counterclockwise rotation of flagella, which produces forward motion. TUMBLE: Clockwise rotation of flagella, which causes bacteria to stay in place. REGULATION: Bacteria goes toward attractant by balancing the relative amounts of tumbles -vs- runs. E. Coli example: ACCOMMODATION: Spontaneous methylation of the MCP leads to deactivation of the MCP. Accommodation resets the cell's sensitivity to the attractants, such that a higher level is required for continued activation. MOLECULAR MEMORY: The overall basis for chemotaxis. The cell senses its level of activation, and can only continue to be activated if it is in a region of increased level of attractants, compared to where it was a short time ago. PHYSICAL BACTERICIDAL AGENTS: HEAT: Wet heat is more effective than dry heat. RADIATION: UV-Light FILTRATION

ANTIBIOTIC DRUGS GENETIC MUTATIONS and REPAIR: Types of Mutations:

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TRANSITION: A mutation exchange of purine for purine or pyrimidine for pyrimidine. CAUSES: UV-Radiation Deaminating and alkylating agents base analogs. Spontaneous TRANSVERSION: A mutation exchange of purine for pyrimidine, or pyrimidine for purine. CAUSES: Strictly spontaneous; no causative agents. MICRO INSERTIONS / MICRO DELETIONS: They can be frameshift mutations, and they can be offset by a corresponding mutation downstream from the original, to restore the reading frame. Missense: Change of coded amino acid from one residue to a different residue. Nonsense: Change of coded amino acid to a stop codon, truncating the protein and rendering it dysfunctional. CONDITIONAL LETHAL MUTATION: A mutation that is lethal under one condition (usually temperature) but not another. This is most frequent with Temperature-Sensitive Mutants. Permissive Condition: The condition under which the bacterium survives. (Low temperature) Non-Permissive Condition: The condition under which the bacterium perishes. (High temperature). MUTAGENS: CHEMICAL MUTAGENS Base Analogs: Compounds that resemble a nucleotide base and cause mispairing when inserted. 5-Bromouracil resembles Thymine and gets in the place of Thymine in a chain, which ultimately can lead to an A--T to G-C transition. Chemical Compounds Intercalating Agents PHYSICAL MUTAGENS Heat: Causes depurination, removes purine from DNA backbone, potentially resulting in deletion. UV-Light: Causes formation of Thymine dimers X-Ray Radiation: Cause double-stranded breaks in DNA that lead to deletions. Can also cause radical formation.

LEARNING OBJECTIVES FOR THIS SECTION

Factors Influencing Bacterial Growth 1. Physical requirements a. Temperature Bacteria have a minimum, optimum, and maximum temperature for growth and can be divided into 3 groups based on their optimum growth temperature: 1. Psychrophiles (def) are cold-loving bacteria. Their optimum growth temperature is between -5C and 15C. They are usually found in the Arctic and Antarctic regions and in streams fed by glaciers. 2. Mesophiles (def) are bacteria that grow best at moderate temperatures. Their optimum growth temperature is between 25C and 45C. Most bacteria are mesophilic and include common soil bacteria and bacteria that live in and on the body. 3. Thermophiles (def) are heat-loving bacteria. Their optimum growth temperature is between 45C and 70C and are comonly found in hot springs and in compost heaps. 4. Hyperthermophiles (def) are bacteria that grow at very high temperatures. Their optimum growth temperature is between 70C and 110C. They are usually members of the Archae and are found growing near hydrothermal vents at great depths in the ocean. b. Oxygen requirements Microorganisms show a great deal of variation in their requirements for gaseous oxygen. Most can be placed in one of the following groups: 1. Obligate aerobes (def) are organisms that grow only in the presence of oxygen. They obtain their energy through aerobic respiration (def). 2. Microaerophiles (def) are organisms that require a low concentration of oxygen (2% to 10%) for growth, but higher concentrations are
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inhibitory. They obtain their energy through aerobic respiration (def). 3. Obligate anaerobes (def) are organisms that grow only in the absense of oxygen and, in fact, are often inhibited or killed by its presense. They obtain their energy through anaerobic respiration (def) or fermentation (def) . 4. Aerotolerant anaerobes (def), like obligate anaerobes, cannot use oxygen to transform energy but can grow in its presence. They obtain energy only by fermentation (def) and are known as obligate fermenters. 5. Facultative anaerobes (def) are organisms that grow with or without oxygen, but generally better with oxygen. They obtain their energy through aerobic respiration (def) if oxygen is present, but use fermentation (def) or anaerobic respiration (def) if it is absent. Most bacteria are facultative anaerobes. c. pH Microorganisms can be placed in one of the following groups based on their optimum pH (def) requirements: 1. Neutrophiles (def) grow best at a pH range of 5 to 8. 2. Acidophiles (def) grow best at a pH below 5.5. 3. Allaliphiles (def) grow best at a pH above 8.5. d. Osmotic pressure Osmosis is the movement of water across a membrane from an area of higher water concentration (lower solute concentration) to lower water concentration (higher solute concentration). No energy is required. To understand osmosis, one must understand what is meant by a solution (def). A solution consists of a solute (def) dissolved in a solvent (def). In terms of osmosis, solute refers to all the molecules or ions dissolved in the water (the solvent). When a solute such as sugar dissolves in water, it forms weak hydrogen bonds with water molecules. While free, unbound water molecules are small enough to pass through membrane pores, water molecules bound to solute are not (see Fig. 1). Therefore, the higher
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the solute concentration, the lower the concentration of free water molecules capable of passing through the membrane. A cell can find itself in one of three environments: isotonic (def), hypertonic (def), or hypotonic (def). (The prefixes iso-, hyper-, and hypo- refer to the solute concentration). In an isotonic environment (see Fig. 2), both the water and solute concentration are the same inside and outside the cell and water goes into and out of the cell at an equal rate. If the environment is hypertonic (see Fig. 3), the water concentration is greater inside the cell while the solute concentration is higher outside. Water goes out of the cell. In an environment that is hypotonic (see Fig. 4), the water concentration is greater outside the cell and the solute concentration is higher inside. Water goes into the cell. Most bacteria require an isotonic environment (def) or a hypotonic environment (def) for optimum growth. Organisms that can grow at relatively high salt concentration (up tp 10%) are said to be osmotolerant (def). Those that require relatively high salt concentrations for growth, like some of the Archea that require sodium chloride concentrations of 20 % or higher halophiles (def). 2. Nutritional requirements In addition to a proper physical environment, microorgaisms also depend on an available source of chemical nutrients. Microorganisms are often grouped according to their energy source and their source of carbon. a. Energy source 1. Phototrophs (def) use radiant energy (light) as their primary energy source. 2. Chemotrophs (def) use the oxidation (def) and reduction (def) of chemical compounds as their primary energy source. b. Carbon source Carbon is the structural backbone of the organic compounds that make up a living cell. Based on their source of carbon bacteria can be classified as autotrophs or heterotrophs. 1. Autotrophs (def): require only carbon dioxide as a carbon source. An autotroph can synthesize organic molecules from inorganic nutrients.
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2. Heterotrophs (def): require organic forms of carbon. A Heterotroph cannot synthesize organic molecules from inorganic nutrients. Combining their nutritional patterns, all organisms in nature can be placed into one of four separate groups: photoautotrophs, photoheterotrophs, chemoautotrophs, and chemoheterotrophs. e. Water f. Growth factors Growth factors are organic compounds such as amino acids (def), purines (def), pyrimidines (def), and vitamins (def) that a cell must have for growth but cannot synthesize itself. Organisms having complex nutritional requirements and needing many growth factors are said to be fastidious (def). Lecture Topic: Bacterial Growth & Nutrition Grow in liquid culture or on solid agar plates - can isolate pure culture by streaking Culture - bacterial population in liquid Inoculum - starting source of culture Rich vs minimal media Minimal - no organics other than C source; have Na, K, Mg, Ca, Fe, NH4, Cl, PO4, SO4 Selective media - identify auxotrophs and antibiotic resistant bacteria Color-indicator plates - identify which sugars can be used as C source MacConkey agar - contains sugar and pH sensitive dye (red in low pH, white in high pH) If add lactose, Lac+ cells grow, ferment lactose, decrease pH and stain colony red. Lac- cells grow, use amino acids as C source, breakdown products include ammonia which increases pH, decolorizes dye and colony is white. Growth curve - lag, log (exponential), and stationary (up to 109 cells/ml) One bacterium in 11 hours can generate 109 cells One bacterium could theoretically multiply to form enough bacteria equal to mass of the earth in less than two days (given unlimited nutritional source) Determining cell density (number of cells per unit volume) 1) Count with microscope - counts both dead and alive 2) Count colonies - each viable cell can form colony because bacteria aren't very motile on agar 3) Measure OD (optical density) Can do this while culture is growing Must calculate standard curve (OD vs cell density) for each bacterial species (because size makes a difference), each growth medium (because bacteria
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change morphology in different media), and each spectrophotometer

Capsule Stain Purpose This stain is a differential stain involving first a negative stain to visualize the capsule surrounding the cell, and a then a counterstain to see the cell. Production of the extracellular capsule contributes to the virulence of certain microbes by making them less vulnerable to phagocytosis. The capsule of Streptococcus mutans contibutes to the formation of dental plaque. Principle The polysaccharide or polypeptide composition of the capsules makes staining difficult. The capsule stain technique stains around the capsule with a negative acidic stain. Then a basic stain will colorize the cell itself. The capsule appears as a white halo between the cell and the darker background. The smear is prepared in the same manner as the negative stain, and the slide is not heat-fixed, since application of heat may destroy or distort the capsule. The negative stain is air-dried before the basic stain is applied.

Capsule stain. The cell is the purple rod in the center of the clear aea. The purple color is from the basic stain, crystal violet. The clear area is the capsule, and the background is colored by the negative, acidic stain (india ink)

Capsule stain, enlarged.

What is luminescence / fluorescence microscopy? The technique involves stimulation of a polished block or thin section by cathode ray (cathodoluminescence; CL) or uv (ultraviolet light at 450-490 nm wavelength), which causes the specimen to luminesce or fluoresce in the visible light spectrum. This visible light is captured in a specially adapted optical microscope. Why should cathodoluminescence be used on reservoir rocks? Many minerals luminesce under cathode rays in different colours, largely due to trace metals contained within the minerals crystal lattice. For example, in carbonates, manganese activates carbonate mineral luminescence, while iron quenches luminescence. In quartz, for example, there are a variety of activators, while aluminium is believed to quench quartz luminescence. Often, subtle changes in trace elements define changes in pore water chemistry during diagenesis. For example increasingly ferroan pore water compositions may result in progressively weaker luminescence zones within individual calcite cement crystals. Similarly, changes in aluminium content may result in zoned syntaxial quartz overgrowths with different luminescence character. How does uv fluorescence differ from cathodoluminescence? Essentially, the two techniques are very similar. However, uv excitation of fluorescence affects different minerals, notably fluorite. Organic materials, most notably hydrocarbons in the form of mobile oil,
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bitumen and dead oil, kerogen and other detrital organic components, also activate uv fluorescence. Therefore, mineral hosts with organic inclusions can be identified, such as micritic matrix in limestones and early diagenetic marine cements. In addition, hydrocarbon inclusions are readily identified within cement crystals, allowing episodes of hydrocarbon migration to be recognised. What are the applications of luminescence/fluorescence microscopy? In carbonate rocks, both cathodoluminescence and uv fluorescence microscopy can be used to:

Identify original depositional fabrics in many dolomitised, neomorphosed and recrystallised fabrics. Recognise cement zonations within blocky calcite and dolomite cements resulting from geochemical and organic fluctuations during diagenesis. Establish cement stratigraphy comparisons between pore systems such as comparison of matrix and fracture cementation histories. In clastic rocks, the technique can be used to: Study carbonate cements within clastic systems in the same way as for carbonate rocks Identify zonations within syntaxial quartz overgrowths and epitaxial feldspar overgrowths, in order to better constrain silicate diagenesis and relate this in a more meaningful way to fluid inclusion and stable isotopic measurements.

Microscopy Seeing the Invisible Metric Measurements o 1 kilometer (km) = 1,000 meters o 1 millimeter (mm) = 10-3 meters o 1 micrometer (mm) = 10-6 meters o 1 nanometer (nm) = 10-9 meters Parts of the Light Microscope 11

Resolution o The ability of the lens to distinguish two points a specified distance apart. o The shorter the wavelength, the greater the resolution. o Best resolution of light microscopes is 0.2 m (2,000X) Refractive Index o Measure of light-bending ability of the medium. o Changing the refractive index of the specimen relative to the medium increases the contrast between the two. o Staining is used to change the refractive index. Immersion Oil o Air has a different refractive index than glass. o Most light would be lost if it had to travel through air between the specimen and the objective lens. o Immersion oil has about the same refractive index as glass and increases the resolving power of the lens. Brightfield Illumination o Usual configuration of the light microscope o Light passes through the specimen and is seen in the eyepiece o Staining gives increased contrast so cells can be seen. Darkfield Illumination o An opaque disk is put between the light and the specimen o Only light refracted by the specimen reaches the eyepiece. o Emphasizes edges of structures against a dark background unstained specimens Phase-Contrast o Light comes through an annular diaphragm (donut) o Direct rays and refracted rays are combined at the eyepiece o Where rays are in phase, see light; where out of phase, see dark. o Used for detailed examination of living cells. o Examples of these forms of microscopy in Fig 3.4 Fluorescence Microscopy o Fluorescence: molecules absorb UV light, emit visible light o Some fluorochromes can be used as stains (FITC and B. anthracis) o Some are attached to antibodies that bind specifically to organisms Electron Microscopy o Uses electron beam instead of light (105 shorter wave length): 2-20nm o Focused with magnets Transmission (TEM):thin section Scanning (SEM): 3D o Specimen dried, stained with metals Staining for Light Microscopy o Simple stains increase contrast o Differential stains differentiate between organisms or structures o Gram stain differentiates between two kinds of cell wall structures o Organisms must be fixed to slide Gram and + Cell Walls 12

Gram Stain Fig 3.10

Acid Fast Stain Spore Stain Capsule Stain What You Should Know o Approximate size of eukaryotic cell, bacterium, and virus o What limits abilioty of microscope to magnify specimen o Advantages of brightfield, darkfield, phase contrast and fluorescence microscopy o Differences between electron microscopy and light microscopy o Purpose and types of information you get from Gram stain, spore stain, and capsule stain

Microbiological Staining Protocols: Cell Structures Stains Summary: Protocols for staining various cell structures, including spores, flagella, and capsules. Refer to Staining Solutions and Reagents page for information on the chemical composition of each stain. Table of contents The Spore Stain The Capsule Stain The Flagella Stain The flagella stain procedure Hanging drop preparation The Cell Wall Stain

The Spore Stain Some bacterial species, such as Bacillus and Clostridium, form spores in response to unfavorable environmental conditions. These spores are resistant to high temperatures, numerous chemicals, ionizing radiation, and extreme desiccation. The spore begins within the bacterial cell (sporangium) as an endospore that occupies a characteristic position within the cell (terminal, subterminal, or central). As development continues, the sporangium eventually disintegrates, leaving a free spore. The size and shape of the spore is unique to each bacterial species and is often useful in identification. The nature of spores makes staining difficult. The primary stain, malachite green, is driven into the cell by heat; however, care must be taken as overheating will cause cells to expel the endospore. Once stained, the spore is relatively hard to decolorize, unlike the rest of the cell, which is counterstained using safranin. Procedure: 1. Prepare and heat-fix a smear of bacterial cultures. (refer to the simple staining protocol). 2. Cover the smear with malachite green staining reagent. 3. Heat the reagent gently by placing the slide on a warm hot plate or by exposing it to the flame of a Bunsen burner. Allow the reagent to steam for 2 to 3 min.; more reagent may be added to prevent drying. 4. Wash the slide with tap water and drain. 5. Cover the smear with safranin for 30 s. 13

Wash the smear with tap water, drain, and blot dry gently prior to microscopic examination. [Top to page]
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The Capsule Stain The cell walls of many bacterial species are surrounded by a polymeric substance referred to as a capsule (if discrete) or a slime later (if amorphous). These capsules perform numerous physiological functions, including conferring resistance to phagocytosis and allowing adhesion to sites with favorable environments. The size of such capsules varies with species and among strains of a species, and may also be affected by the composition of the growth medium. Because most capsule materials are water soluble, simple stains will not adhere to them, thus making capsule staining a difficult process. As a result of this limitation, capsule staining works by staining the bacteria and background more intensely than the capsule itself. Capsule staining is diagnostically useful since the presence of large capsules generally indicates the presence of a virulent form of some bacterial species (e.g. pneumococci); by forcing capsular swelling (e.g. through the exposure the certain anti-bodies), a condition known as the Quellung phenomenon, the strain of bacteria can be easily characterized. Procedure: 1. Add a small amount of bacterial culture to a drop of nigrosin solution on one end of a slide. Mix without spreading. 2. Spread the mixture over the slide using the end of a second slide to prepare a thin smear as in a negative stain. 3. Let the smear air dry. 4. Fix the smear with absolute alcohol for 2 min; drain off the alcohol and let the smear dry. 5. Stain with crystal violet for 2 min. 6. Wash the slide with tap water, and air dry vertically before microscopic examination. The Flagella Stain Motile bacteria, particularly eubacteria (true bacteria), possess one or more flagella. Flagella are coiled in rigid spirals that revolve around their points of attachment; however, due to the nature of microscopic preparation, flagella are seen as "flat", wave-like organelles. Further, though flagella are many times the length of the bacterial cell, they are about 100 times narrower than most bacterial cells, averaging 0.01 to 0.05 micrometers (m); hence, flagella are beyond the resolving power of light microscopes and can be seen directly only with an electron microscope. Because electron microscopes are usually not readily available, staining allows the visualization of flagella using light microscopy: the thickness of the flagella is increased by coating them with mordants and then staining them; the staining procedure must be carried out with care as rough handling may cause the flagella to break off. Like all other cell structure stains, the flagella stain aids in the identification of unknown bacteria. Flagella stain procedure 1. Obtain new slides and rinse them in 95% ethyl alcohol. Wipe them dry with lintless paper tissue, and pass them through the flame of a Bunsen burner several slides. Extremely clean slides are essential when preparing a flagella stain 14

Prepare a light suspension of bacterial culture in distilled water, and incubate for 10 to 15 min to develop and extend the flagella fully. Handle the suspension gently so that the flagella will not break off. 3. Transfer one loopful of the suspension of cells to one end of a clean slide. Tilt the slide, allowing the drop to run down. Air dry this film; do not heat-fix. 4. Flood the slide with flagella mordant, and allow to stand for approximately 10 min. 5. Rinse the stain off gently with distilled water. 6. Flood with Ziehl's carbol fuchsin for 5 min. Rinse off gently with water. 7. Air dry (do not blot) before microscopic examination. Hanging drop preparation: allows for the detection of motility under a microscope 1. Ring the outer edge of the concave well of a depression slide with a small amount of petroleum jelly. (Plain slides may also be used by building up a circle of petroleum jelly smaller than the cover slip.) 2. Place a small drop of bacterial suspension on the cover slip, and then invert the slide wide the concave area over the drop. In a properly made preparation, the drop will hang from the cover slip; the petroleum jelly forms a seal and prevents rapid evaporation of the drop. 3. When observing the specimen, use the high power objective, exercising care in focusing and adjusting the light. Proper illumination is critical as this is an unstained preparation. Concentrate on the edge of the droplet, where the organisms will be most active because of a greater oxygen supply.
2.

The Cell Wall Stain The rigid cell walls present in most bacteria are difficult to identify with a light microscope because they look similar to other cell components. However, the use of special reagents allow the wall to be stained and observed. The normally negative bacterial cell surface is made positive by treating it with a cationic surface agent, such as cetylpyridinium chloride; the now positive cell surface now takes on the negatively charged chromophore of an acidic dye. The bacteria is then counterstained prior to visualization. Under the bright field microscope, the cell walls appear blue while the bacterial cytoplasm appears red. The cell wall stain indicates the presence of cell walls (though not their composition) and is particularly useful in identifying bacterial species that lack a cell wall, e.g. mycoplasms. Procedure: 1. Prepare and heat-fix a smear of bacterial cultures. (refer to the simple staining protocol). 2. Add 3 drops of cetylpyridinium chloride solution to each smear. 3. Add 1 drop congo red to each smear. Mix the drops with a inoculating loop. Use caution to avoid scratching the smears. 4. Rotate the drops with a tilting motion by holding the slide in your hand. 5. Rinse off with tap water. 6. Counterstain for 10 s. with methylene blue. 7. Rinse and blot dry prior to microscopic examination.

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