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MICROBIOLOGY: THE SCIENCE

MICROBIOLOGY the study of very small living organisms organisms called microorganisms or microbes
(viruses, bacteria, some algae, protozoa, and some fungi).
Branches of Microbiology
1. Bacteriology study of bacteria
2. Virology study of viruses
3. Phycology study of algae
4. Protozoology study of protozoans
5. Mycology study of fungi
Why Study Microbiology?
1. Indigenous microflora/normal flora
2. Opportunistic pathogens organisms that cause disease
3. Microorganisms contribute more oxygen to our atmosphere than do plants (algae/cyanobacteria)
4. Saprophytes or decomposers (nitrates/phosphates)
5. Bioremediation (decomposition of industrial waste).
6. Microbial ecology the study of the relationships between microbes and the environment
7. Microbes serve as important links in food chains.
8. Microbes living in the gut of animals aid in digestion or produce substances of value to the host animal.
9. Biotechnology the use of microbes in industry (food and beverage).
10. Some bacteria and fungi produce antibiotics
11. Genetic engineering
12. Microbes are used as cell models
13. Microbes cause 2 categories of disease:
a. Infectious disease results when a pathogen colonizes the body and subsequently causes disease
Pathogens microorganisms that cause disease (about 3% of known microbes)
Nonpathogens microorganisms that do not cause disease
b. Microbial intoxication results when a person ingests a toxin (poisonous substance) that has been
produced by a microorganism.
PIONEERS IN THE SCIENCE OF MICROBIOLOGY
Anton Van Leeuwenhoek (1632-1723)
First person to see live bacteria and protozoa (animalcules)
Father of Microbiology, Father of Bacteriology, Father of Protozoology
An amateur scientist who recorded his observations and sent them to the Royal Society of London.
Theory of Spontaneous Generation or Abiogenesis
This theory arose from the idea that life can arise spontaneously from nonliving material.

Following the work of others, Louis Pasteur and John Tyndall finally disproved this theory.
Rudolf Virchow introduced the theory of biogenesis.

Louis Pasteur (1822-1895)


Demonstrated that different kinds of microorganisms produce different fermentation products
Made experiments to disprove the theory of spontaneous generation
Developed Pasteurization, a process to kill pathogens in many types of liquid
Discovered the causative agent of silkworm disease; introduced the terms aerobes, anaerobes
Made significant contributions to the germ theory of disease the theory that specific microorganisms
cause specific infectious disease
Developed vaccines to animal diseases including rabies which later was used to treat human rabies
Robert Koch
Discovered that Bacillus anthracis produces spores
Developed methods of fixing, staining, photographing bacteria, and solid bacterial culture media
Discovered Mycobacterium tuberculosis, the causative agent of TB and Vibrio cholerae, the agent of
cholera
Introduced Kochs Postulates and the concept that a specific microbe is the cause of a specific disease.

Kochs Postulates
a) the microbes should be invariably present in the diseased individuals
b) the microbes/etiologic agent should be isolated from the diseases individuals & grown in pure
culture.
c) Inoculation of the agent/microbe into a healthy experimental animal should produce disease similar
to that observed initially.
d) the presumed pathogen should be isolated from the animal with experimentally induced disease

CELL STRUCTURE AND TAXONOMY


Cell the fundamental unit of any organism
Cellular structure of microorganisms
1. Acellular Viroids, prions, and viruses
2. Cellular
a. Prokaryotes Archaea, bacteria, cyanobacteria
b. Eukaryotes Algae, protozoa, fungi
Comparison Between Eukaryotic and Prokaryotic Cells
Eukaryotes
-larger ribosome (80s)
-possess a true nucleus with nuclear membrane
-compartmentalized (membrane-bound organelles)

Prokaryotes
-smaller ribosome (70s)
-noncompartmentalize

Eukaryotic cell structure


A. Cytoplasmic structure
1. nucleus contains the DNA
2. nucleolus site of ribosomal RNA synthesis
3. endoplasmic reticulum (ER) rough, smooth
4. golgi apparatus
5. ribosomes site of protein synthesis, attached to ER, 80s in size and dissociates into 60s
and 40s
6. mitochondria energy production, powerhouse
7. lysosomes hydrolytic enzymes
8. chloroplast photosynthesis
B. Cell envelop structures
1. plasma membrane regulates transport into and out of the cell, bilayered lipoprotein, with
sterols
2. cell wall provides rigidity and strength
3. motility organelles
a. cilia shorter projections
b. flagella longer
Prokaryotic Cell Structure

Prokaryotic cells are about 10 times smaller than eukaryotic cells.

Reproduction of prokaryotic cells is by binary fission the simple division of one cell into two cells,
after DNA replication and the formation of a separating membrane.
Parts of a prokaryotic cell:
A. Cytoplasmic Structure
1. nucleoid DNA appears as diffuse nucleoid, not membrane-bound
-DNA is single, circular chromosome of double stranded DNA
*plasmids extrachromosomal circular genetic elements, double stranded DNA
-contain antibiotic resistance genes-infections difficult to treat
2. ribosomes involved in the translation process during CHON synthesis
-free in the cytoplasm
-70s in size and dissociates into 50s and 30 s subunit
3. cytoplasmic granules/metachromatic granules storage deposits

4. Spores (endospores) dormant forms of bacteria that are resistant to heat (boiling), cold, drying &
chemical agents
-destroyed by autoclaving (Bacillus stearothermophilus-indicator organism)
-produced only by Bacilllus (gram-positive, aerobic) and Clostridium (Gram-positive, anaerobic)
-used as a means of survival when their moisture or nutrient supply is low
-in the laboratory, bacteria can be induce to form spores
Endospores multilayered protective coat consist of:
a. cell membrane
b. thick peptidoglycan mesh
c. another cell membrane
d. a keratin-like CHON wall
e. an outer layer called exosporium
B. Cell envelope structures
1. Cell membrane regulates transport across the membrane
2. Cell wall (Murein layer) rigid structure, composed of alternating units of disaccharide Nacetylglucosamine (NAG) and N-acetylmuramic acid (NAM) (crosslinked)
-different types for G+, G-, acid-fast
Gram-positive-thick peptidoglycan, teichoic, lipoteichoiic acid (unique to gram-positive)
-other gram-positive bacteria (e.g. mycobacteria) have waxy substance called mycolic acid that make
their cells refractory or resistant to toxic substances including acids
Gram-negative more complex, thinner peptidoglycan layer, has lipopolysaccharide (LPS), an
endotoxin that is an important virulence factor. An endotoxin causes shock, sepsis, DIC, and
leukopenia
3. Glycocalyx
a.Slime Layer enable bacteria to glide or slide along solid surfaces
b. Capsule serve an antiphagocytic function, a virulence factor
2 important test to visualize capsules under the microscope & aid in bacterial identification
*India ink not taken up by the capsule, capsule appears as a transparent halo around the
cell (used for the ID of Cryptococcus neoformans)
*Quellung reaction when antibody binds to bacteria, the capsule swells and this can be
visualized microscopically
-Capsule can be removed by boiling a suspension of the microorganism, somatic or cell wall
antigens can be detected by serotyping
4. cell appendages
a. Pili (Fimbriae) hairlike structures most often observed on Gram negative bacteria; composed
of pilin; are thinner than flagella and not associated with motility
* for attachment quite numerous
e.g. N. gonorrheae has pili that bind to cervical cells & buccal cells to cause gonorrhea. E. coli
and Campylobacter jejuni cant cause diarrhea w/o binding to the intestinal epithelium. Bordetella
pertussis binds to ciliated respiratory cells & cause whooping cough)
*Sex Pilus for transfer of genetic material (conjugation)
b. flagella
Atrichous no flagella (e.g. Shigella)
Peritrichous flagella over entire surface (E. coli, Proteus mirabilis)
Lophotrichous two or more flagella at one end
Amphitrichous single flagella at each end
Monotrichous single polar flagellum (Vibrio cholera)
Axial filaments two flagella-like fibrils attached to each end of the bacterium (Spirochetes)
Bacterial Taxonomy

Taxonomy (the science of classification of living organisms) consists of three separate but interrelated
areas: classification, nomenclature, and identification.

Classification is the arrangement of organisms into taxonomic groups on the basis of similarities or
relationships. (Kingdoms/Domains, Divisions/Phyla, Classes, Orders, Families, Genera, and Species)

Nomenclature is the assignment of names to the various taxa according to international rules.

Identification is the process of determining whether an isolate belongs to one of the established,
named taxa or represents a previously unidentified species.

The binomial system of nomenclature developed by Carolus Linnaeus in the 18 th century assigned
two names to each organism. The first name is the genus and the second name is the specific epithet.

The first and second names together are referred to as the species. Ex. Escherichia coli, Pseudomonas
aeruginosa.

DOMAIN BACTERIA
CHARATERISTICS OF BACTERIA
Cell Morphology

Size ranges from spheres of 0.2 m in diameter to 10 m long spiral shaped bacteria to even longer
filamentous bacteria.

Basic Shapes of Bacteria


1. Cocci round or spherical ex. Enterococcus spp., Neisseria spp., Staphylococcus spp., and
Streptococcus spp.
Diplococci pairs
Streptococci chains
Staphylococci clusters
Tetrads packets of four
Octads packets of eight
2. Bacilli rectangular or rod-shaped; may be short or long, thick or thin, and pointed or with curved or
blunt ends. (ex. Family Enterobacteriaceae Enterobacter, Escherichia, Klebsiella, Proteus,
Salmonella, and Shigella spp., Pseudomonas aeruginosa, Bacillus spp., and Clostridium spp.)
Diplobacilli pairs
Streptobacilli chains
Coccobacilli short rods resembling elongated cocci (Listeria monocytogenes and Haemophilus
influenza)
Palisade arrangement bacteria stack up next to each other, side-by-side; characteristic of
Corynebacterium diphtheriae (diphtheroids)
3. Curve and spiral-shaped bacteria
Curve comma-shaped. Ex. Vibrio spp.
Gull-wing morphology a pair of curved bacilli. Ex. Campylobacter spp.
Spiral referred to as spirochetes (tightly coiled ex. Treponema pallidum; less coiled ex.
Borrelia spp.
4. Pleomorphic lacking a distinct shape
Pleomorphism the ability of some bacteria to exist in a variety of shapes (Mycoplasma no
cell wall)
STAINING the process of artificially coloring the microorganism with dyes for the following purposes:
1. To observe and appreciate the appearance of microorganisms.
2. To differentiate microorganisms
3. For the demonstration of some special structures (capsule, cell wall, flagella)

Different Staining Methods


Simple Staining uses only one dye: methylene blue blue; carbol fuchsin red
Differential Staining makes use of 2 or more dyes. Microorganisms are stained
according to a particular dye which they have an affinity with (Ex. G/S, AFS M. tuberculosis).
3.
Special Staining uses dyes which stain special structures of the microorganism
capsules, spores, flagella (Ex. India Ink)
1.
2.

GRAM STAIN separates bacteria into 2 groups: Gram-positive and Gram-negative


Crystal Violet primary stain (blue stain)
Grams Iodine mordant
95% alcohol decolorizer
Safranin Red secondary/counter stain (red stain)

Preparation of a Smear for Any Staining Procedure


Step 1: Make a smear. Let the smear air dry before proceeding.
Step 2: Fix the smear by methanol- or heat-fixation. The smear is now ready for Gram staining.

Gram Staining Procedure (VIAS)


1. Flood smear with crystal violet (primary stain) for 1 minute. Wash off stain with water.
2. Flood smear with iodine (mordant) for another minute. Wash off stain with water.
3. Decolorize with acetone alcohol for about 1-3 sec (follow manufacturers instruction)
- very critical step
4. Counterstain with safranin for 30 seconds.
5. Wash off stain with water.
Differences Between Gram-Positive and Gram-Negative Bacteria
Gram-Positive
Gram-Negative
Bacteria
Bacteria
Color at the end of the Gram staining
procedure
Blue-to-purple
Pink-to-Red
Peptidoglycan in cell walls
Thick layer
Thin layer
Teichoic acids and lipoteichoic acids in cell
walls
Present
Absent
Lipopolysaccharide in cell walls
Absent
Present
All cocci are gram-positive except, Neisseria, Veilonella, Branhamella
All bacilli are gram-negative except Bacillus, Clostridium, Corynebacterium, Erysephilothrix, Lactobacillus,
Listeria, Bifidobacterium, Eubacterium and Mycobacteria
Gram-variable bacteria: members of the genus Mycoplasma/Mycobacterium.
ACID-FAST STAIN (CAM) procedure used to detect organisms that do not stain well with
conventional stains (e.g. Mycobacterium spp., Nocardia & Actinomyces). These organisms have high
lipid content in their cell walls. Once stained, they are very resistant to decolorization by acid alcohol
1. Flood smear with carbol fuchsin (primary stain) and heat until steaming of the stain occurs. (Heat
drives the stain into the cell wall). Leave for 5 minutes (always follow manufacturers instruction)
2. Decolorize with acid-alcohol (3% HCl) until no more stain comes off.
3. Counterstain with methylene blue (counterstain/secondary stain)
4. Wash off stain with distilled water (presence of nonpathogenic Mycobacteria).
Acid-fast organisms appear red in a background of blue or green.
Ziehl-Neelsen hot method
Kinyoun stain cold method (uses malachite green as counterstain), no heating because the stain is
stronger/more concentrated

Bacterial Motility associated with the presence of flagella or axial filaments

Most spiral-shaped bacteria and about one-half of the bacilli are motile

cocci are generally nonmotile

Motility demonstration: flagella stain, stabbing bacteria in semisolid medium, using the
hanging-drop technique.
Colony Morphology includes size, color, overall shape, elevation, & appearance of the edge or margin
Atmospheric Requirements in the microbiology laboratory it is useful to classify bacteria on the basis of
their relationship to oxygen and carbon dioxide.
Nutritional Requirements all bacteria need some form of the elements carbon, hydrogen, oxygen, sulfur,
phosphorus, and nitrogen for growth. Certain microbes have specific vitamin requirements and some need
organic substances secreted by other living microbes during their growth; they are said to be fastidious
Biochemical and Metabolic Activities as bacteria grow, they produce many waste products and
secretions, some of which are enzymes that enable them to invade their host and cause disease; pathogenic
bacteria can be tentatively identified by the enzymes they secrete.
Genetic Composition most laboratories are moving toward the identification of bacteria using some type of
test procedure that analyzes the microbes DNA or RNA.