The Study of Microbial Structure: Microscopy and Specimen Preparation Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation

Measurement of microbes – units known as micrometers. 1000 micrometers = 1 millimeter Length of bacteria – 2 um to 7 um Diameter 0.2 um to 2 um Bright field microscope Total magnification = magnification by the objective lens X magnification by the ocular lens Resolving power (resolution) clarity/sharpness of the image

The Light Microscope many types bright-field microscope dark-field microscope phase-contrast microscope fluorescence microscope

The Bright-Field Microscope produces a dark image against a brighter background has several objective lenses parfocal microscopes remain in focus when objectives are changed Compound microscope- ocular lens and objective lens total magnification product of the magnifications of the ocular lenses and the objective lenses

Figure 2.3

Microscope Resolution ability of a lens to separate or distinguish small objects that are close together Resolving power of the microscope in our lab 0.2 um 1000 um = 1 mm 1 um = 1/1000 mm

The Dark-Field Microscope produces a bright image of the object against a dark background used to observe living, unstained preparations For eucaryotes has been used to observe internal structures For procaryotes has been used to identify bacteria such as Treponema pallidum, the causative agent of syphilis

Fig. 2.8a

The Phase-Contrast Microscope enhances the contrast between intracellular structures excellent way to observe living cells Especially useful for detecting bacterial components such as endospores and inclusion bodies

Fig. 2.8d endspores

The Fluorescence Microscope exposes specimen to ultraviolet, violet, or blue light specimens usually stained with fluorochromes auramine O shows a bright image of the object resulting from the fluorescent light emitted by the specimen Has applications in medical microbiology Mycobacterium tuberculosis

Fig. 2.13b Mycobacterium

Figure 2.18

Beam of electrons is used in place of light. TEM - thin sections of the specimen are obtained and placed on a copper mesh grid. Magnifies the object 10,000X to 100,000X. It has the resolving power of .0025 um. This used to observe internal structures. SEM – used to observe structures found on the surface of microbes. Magnifies the object 1000X to 10,000X. Resolving power of 0.02 um.

Fig. 2.17b TEM Rhodospirillum 100,000X

Fig. 2.17c TEM T4 bacteriophage

Fig. 2.24a 32,000X

Preparation and Staining of Specimens increases visibility of specimen accentuates specific morphological features (shape, arrangement) preserves specimens

Dyes and Simple Staining make internal and external structures of cell more visible by increasing contrast with background ability to bind cells (ionic bond is formed between the cell and the dye)

Dyes and Simple Staining Ionizable dyes have charged groups basic dyes have positive charges acid dyes have negative charges simple stains a single stain is used use can determine size, shape and arrangement of bacteria

Dyes are salts Basic dye – positive ion has the color. Methylene blue chloride. Acidic dye – negative ion has the color. Sodium eosinate Basic dyes are used to stain the cell. Bacterial cell is negatively charged. It is attracted to the positive ions. Ionic bond is formed between the cell and the stain.

Negative cell is repelled by the negative ions Negative cell is repelled by the negative ions. They are used to stain the background. Nigrosin is used – background is black and cells are bright. Image is similar to what is seen in the case of dark-field. (negative staining) Simple staining – a basic dye is used to stain the cell to determine the shape and arrangement of the cells. Gram staining is a differential staining. It places bacteria into 2 groups.

Fig. 2.14a

Differential Staining divides microorganisms into groups based on their staining properties e.g., Gram stain e.g., acid-fast stain

Gram staining most widely used differential staining procedure divides bacteria into two groups based on differences in cell wall structure

Gram staining Crystal violet – primary stain Iodine – mordant Alcohol-acetone - decolorizer Safranin – counterstain Gram + are purple Gram – are pink

Figured 2.15

Gram staining is based on the cell wall structure. Gram positive cells have thick cell walls. They hold on to the primary stain. Gram negative cells have thin cell wall. One or two layers of peptidoglycan. They also have an outer membrane – lipids. Alcohol causes damage to the lipids. Primary stain leaks out.

Acid-fast staining particularly useful for staining members of the genus Mycobacterium e.g., Mycobacterium tuberculosis – causes tuberculosis e.g., Mycobacterium leprae – causes leprosy high lipid (mycolic acid) content in cell walls is responsible for their staining characteristics

Acid-fast staining Differential staining Two genera are acid-fast Mycobacterium and Nocardia They have a waxy substance known as mycolic acid in their cell walls.

Carbolfuchsin – primary stain Acid-alcohol – decolorizer Methylene blue – counterstain Acid-fast – red Nonacid-fast - blue

Fig. 2.14d

Capsule staining A capsule is a gelatinous substance found around the cell wall. It cannot be stained Stain the background using nigrosin. Stain the cell with crystal violet. Background is black. Capsule shows up as a clear ring around the stained cell.

Staining Specific Structures endospore staining double staining technique bacterial endospore is one color and vegetative cell is a different color flagella staining mordant applied to increase thickness of flagella

Endospore staining Two common genera of bacteria that make endospores are Bacillus and Clostridium. Endospores are resistant to hostile environmental conditions. Heat, UV light, disinfectant, desiccation. Endospores are formed within the vegetative cell. Once the formation is complete, endospores are released into the environment.

Endospore Staining Malachite green - primary stain Water – decolorizer Safranin – counter stain Endospores – green Vegetative cells -pink

Fig. 2.14g Tannic acid – increases thickness fuchsin

Beam of electrons is used in place of light. TEM - thin sections of the specimen are obtained and placed on a copper mesh grid. Magnifies the object 10,000X to 100,000X. It has the resolving power of .0025 um. This used to observe internal structures. SEM – used to observe structures found on the surface of microbes. Magnifies the object 1000X to 10,000X. Resolving power of 0.02 um.