Microbiology and Immunology
General Microbiology
Microorganisms
General Considerations
A Ubiquitous—found virtually everywhere
B Only 3% are pathogenic (disease causing); 97% are nonpathogenic
C Exhibit characteristics common to all biologic systems: reproduction, metabolism, growth, response to stimuli, adaptability, mutation, and organization
D Medically important microorganisms
a. Protozoa—unicellular, nonphotosynthetic, heterotrophic
2. Prokaryotes (aerobic or anaerobic unicellular bacteria)
3. Viruses; classification based on:
a. Type and properties of nucleic acid
b. Morphology of nucleoproteins
c. Presence and properties of envelopes—the envelope is the protein coat that protects the capsid and nucleic acid of the virus
a. Type of protein found in brain neurons; contains no genetic material
b. Not bacterial, viral, or fungal
c. Improper folding and inability to be degraded allows for accumulation and damage to the nervous system
d. Transmissible (spongiform encephalopathies)
e. Highly resistant to traditional sterilization methods because of the extreme stability of prion proteins
Methods of Measurement and Observation
1. Macroscopic: measurable and observable by the naked eye
2. Microsocopic: too small to be measured or observed by the naked eye. Requires a microscope or lens to see
B Most commonly used units of measurement
C Light microscopes illuminate objects by visible light
a. Used to observe the morphologies of microorganisms
c. Cannot be used to observe microorganisms <0.2 µm, such as viruses and spirochetes
a. Specimens seen as bright objects against a dark background
b. Used for the examination of unstained microorganisms and spirochetes and hanging-drop preparations
c. Advantage—allows a view of living bacteria not visible by Gram stain; undisturbed in size or shape by fixing and staining techniques
a. Useful in examining transparent, living cells, including their internal structure, and in determining motility in a fluid medium; can show dense structures
b. Variations in density between the microbes and the surrounding medium are capitalized on to increase the contrast between the two
a. Used to visualize objects that fluoresce or emit light when exposed to light of different wavelengths
b. Ultraviolet light, fluorescent chemicals, and special filter systems required
c. Commonly used in the medical field to track antigen–antibody reactions and as a diagnostic technique (immunofluorescence)
5. Confocal-scanning laser microscopy
a. A conventional light microscope uses a laser light source to illuminate planes of a fluorochrome-stained specimen
b. Confocal images combine fluorescent and reflected images
c. Successive planes are scanned until the entire specimen is scanned
d. Useful in creating three-dimensional pictures of biofilms
(a) Thin films of microorganisms are spread on a glass slide and allowed to dry (smear)
(b) Films are fixed, either by a chemical fixative or by passing through a flame; this denatures the proteins and kills the cell
(c) Dyes or stains are applied to the smear to allow for greater visualization; allows for some differentiation of species
(d) Fixation process tends to reduce the sizes of cells; dye addition tends to increase the sizes of cells
(a) Acidic, or negative, dye is used to stain basic components of the cell (e.g., glycoproteins, matrix)
(b) Basic, or positive, dye is used to stain acidic components of the cell (e.g., nucleic acid and polysaccharides)
(3) Simple staining procedures
(a) Use a single dye (e.g., carbolfuchsin, crystal violet, methylene blue, or safranin)
(b) Are used to show shapes, sizes, and arrangements of bacterial cells
(4) Differential staining procedures (Table 9-1)
TABLE 9-1
Comparison of Gram-Positive and Gram-Negative Bacteria
| Gram-Positive Bacteria | Gram-Negative Bacteria | |
| Color after Gram’s stain procedure | Blue to purple | Pink to red |
| Peptidoglycan layer in cell walls | Thick | Thin |
| Teichoic acid in cell walls | Present | Absent |
| Lipopolysaccharide in cell walls | Absent | Present |
(a) More than one dye preparation used
(b) Used for initial bacterial grouping
[1] Gram stain—differentiates microorganisms based on color as gram positive (blue to purple) or gram negative (pink to red); certain characteristics of microorganisms appear correlated with their staining reactions: cell wall thickness, chemical composition, and sensitivity to penicillin; useful in the diagnosis of infectious diseases
[2] Acid-fast stain—differentiates between acid-fast and non–acid-fast bacteria; differentiates mycobacteria (e.g., Mycobacterium leprae and Mycobacterium tuberculosis) from other bacteria by indicating the presence or absence of special lipids in the cell wall; organisms resist decolorization with an acidic solution of alcohol after being stained with a basic dye
(5) Special staining procedures—used to color and isolate specific parts of microorganisms
(a) Negative staining for capsules—determines if organism is encapsulated
(b) Schaeffer-Fulton spore stain (e.g., Bacillus, Clostridium)—determines if organism is a spore former
(c) Flagellar staining—determines if organism has flagella
(d) Toluidine blue-O staining—determines prions, proteoglycans, and glycosaminoglycans in tissues
Prokaryotic (Bacterial) Cell Structure and Function
b. Occur in pairs (diplococci), chains (streptococci), four-in-a-square arrangement (tetrad), eight cells in a cubic arrangement (sarcinae), and irregular clusters (staphylococci)
2. Bacilli (singular, bacillus)
b. Occur in pairs (diplobacilli); chains (streptobacilli); small, rounded rods (coccobacilli); and with tapered ends (fusiform bacilli)
3. Spirilla (singular, spirillum)
4. Palisade arrangement (bacterial cells form weird angles to one another)
(1) Flagella (singular, flagellum)
(a) Threads of protein that extend from the cell surface and move in a whip-like motion
(b) Vectored motility must be distinguished from brownian movement, which is caused by bacteria randomly hitting molecules in the surrounding medium; flagella enable bacteria to move toward favorable environments and away from adverse ones (chemotaxis)
b. Allow for movement—spirochetes (e.g., Treponema pallidum, Borrelia burgdorferi) move by this method
2. Surface coating (glycocalyx)
(1) Condensed and well-defined masses of polysaccharides or polypeptides firmly attached to the cell wall
(2) Encapsulation protects pathogenic organisms from drugs, phagocytosis, and bactericidal factors
(3) Some bacteria need capsules to maintain virulence (e.g., Streptococcus pneumoniae, Streptococcus mutans)
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