Introduction to Microbiology

Bacteriology

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Microbiology : Study of Microorganisms Cellular organisms Bacteria Parasites Fungi Acellular particles Virus Prions

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First type of cells on earth Prokaryotes 2 Billion yrs. In 1980 Woese defined the three cellular Domains

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Domains

Archea

Prokaryotes
Bacteria

Protists ( Algae, protozoa) Fungi plants Animals

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Archaea
Archaea different from Bacteria Lives mostly harsh environments No known pathogens or parasites Methanogens are used in biogas production and sewage treatment

Archaea were first found in extreme environments, such as volcanic hot springs.

Methanobrevibacter smithii in human gut

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Archaea recently discovered in acid mine drainage.

Bacteria
Prokaryotes- No Nuclear Membrane And Cellular Organelles
Lack Sterols In Cell Membrane

Circular DNA
Ribosomes 70S ( 30S & 50S )

1micron In Size

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Bacteria & Archaebacteria difference

Bacteria
Peptidoglycan (Murein)

Muramic Acid
16S rRNA sequence different

Archaebacteria
Pseudomurein

No Muramic Acid
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Major Differences between domains:

Cell configuration Eukaryotes eucaryotic Bacteria procaryotic Archaea procaryotic

Nuclear membrane Present Chromosome shape linear Cell membrane sterols present

Absent circular absent

Absent circular absent

Ribosome size
Organelles (mitochondria and chloroplasts) Amino acid initiating protein synthesis

80S (cytoplasmic)
present

70S
absent

70S
absent

methionine

N-formyl methionine

methionine

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In 2001 Bergey's Manual of Systematic Bacteriology has established 24 phyla of bacteria, systematically ordered into class, order, family, genus and species. For example, E. coli is in the
Domain Bacteria, Phylum Proteobacteria, Class Gamma Proteobacteria, Order Enterobacteriales, Family Enterobacteriaceae, Genus Escherichia, Species E. coli.
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 1665 Robert Hooke observed living plant tissues (20X mag.)

Little boxes or Cells

Used simple magnifying lens Suggested all living things are made of cells

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Hooke's Microscope

1665

Antonie van Leeuwenhoek was inspired by this publication

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 Observed
Bacteria Protozoa Sperm cells Blood cells Microscopic worms

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Louis Pasteur (1822-1895)

Father of microbiology Fermentation (1857) Pasteurization: heat liquid enough to kill spoilage bacteria (1864) Vaccine development rabies Proposed the germ theory of disease

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Robert Koch, (1876)

Postulates Germ theory (1876) Identified microbes that caused anthrax (1876), tuberculosis (1882) and cholera (1883) Developed microbiological media & streak plates for pure culture (1881)

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Kochs postulates
1) Specific microorganism is present in all cases of the disease 2) Organism can be obtained in pure culture outside of the host 3) Organism when re-inoculated into host causes the same symptoms

4) Organism can be isolated in pure culture from

experimentally infected host
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Bacteria need Five Essential Structural Components:

A Nucleoid (DNA),

Ribosomes,
Cell Membrane, Cell Wall, And Some Sort Of Surface Layer, Which May Or May Not Be An Inherent Part Of The Wall

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Bacterial Structure
Pili or Fimbriae DNA

Cell Membrane Cell wall Capsule or Slime layer

Inclusions Ribosomes

Plasmid

Flagella Mesosomes
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Drawing of a typical bacterial cell, by Vaike Haas, University of Wisconsin-Madison

Courtesy: Electron micrograph of Streptococcus pyogenes by Maria Fazio Dr.Aravind and Vincent A. Fischetti, Ph.D

Bacterial components
Cell surface appendages:
Flagella Pili or fimbrea

Cell envelop:

Capsule & Slime layer Cell wall ( Peptidoglycan ) Cell membrane

Cytoplasm:
Nucleoid ( Circular DNA ) Ribosomes 70s Plasmids Mesosomes Inclusions & etc
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Flagella:
Motility Attached to Cell membrane

Powered by proton motive force (chemiosmotic potential)

Antigenic Observed by fluid wet mount media

Types:
a) b) c) d) Monotrichous ex: vibrio cholera Peritrichous ex:E.coli Lopotichous ex:helicobacter Amphitrichous ex:rhodosprillum rubrun
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Courtesy by Nature Group

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Fimbriae and pili: Interchangeable terms

Hair-like structures Involved in adherence and colonization Inhibits Phagocytosis ex: Niserria gonococci, E.coli, M protein & Pili S.pyogenes. Antigenic F or sex pilus conjugation ex: E.coli

Pili

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Courtesy by Dr. Kenneth Todar

Capsule:
Discrete detectable layer of polysaccharides deposited outside the cell wall is Capsule A less discrete polysaccharide or matrix which embeds the cells is called as slime layer or a biofilm. Antigenic Inhibits Phagocytosis & desiccation

Dental plaque biofilm protected from environment for themselves and other bacteria
Capsule - Indian ink and qulung reaction Not slime layer Meningitis Polysaccharides, hyaluronic acid & D- glutamic acid

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hyaluronic acid capsule that surrounds the exterior of the bacteria

Bacterial capsules outlined by India ink viewed by light microscopy. This is a true capsule, a discrete layer of polysaccharide surrounding the cells.
Courtesy: Electron micrograph of Streptococcus pyogenes by Maria Fazio and Vincent A. Fischetti, Ph.D.
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Dental plaque revealed by a harmless red dye. Colonies of Bacillus anthracis. The slimy or mucoid appearance of a bacterial colony is usually evidence of capsule production.

Courtesy: micrograph by Marilee Sellers, Northern Arizona University

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Courtesy by Dr.Kenneth Todar

Cell wall
Rigid wall inhibits osmatic rupture

Murein is a unique type of peptidoglycan

Disaccharides (glycan) cross-linked by short chains of amino acids (peptide) = Peptidoglycan Peptidoglycan layer is done with periplasmic enzymes of bacteria called transglycosylases, transpeptidases and carboxypeptidases The beta lactim drugs prevent the action of these enzymes and bacterial growth is inhibited
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 Peptidoglycan is made up of
2 sugars (Glycans):
N-acetyl-glucosamine = G N- acetylmuramic acid = N

4 amino acids (Peptides):

L-alanine = L-ala D-glutamic acid = D-glu diaminopimelic acid = DAP D-alanine = D-ala

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G N-acetyl glucosamine N N-acetyl Muramic acid

Bacterial cell wall

L-alanine = L-ala D-glutamic acid = D-glu diaminopimelic acid = DAP D-alanine = D-ala

Peptidoglycan

Amino acid side chain

Transpeptidases and Carboxypeptidases bonds 2 tetra peptide chains

Tetra peptide ( 4 Amino acids ) bridges

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Action of beta lactam drugs and Lysosomes

*Pencillin inhibits Transpeptidases and Carboxypeptidases function

X
Lysosomes cleaves bond between N- acetyl glucosamine and N- acetyl Dr.Aravind muramic acid

 2 types of cell walls:

Gram + (Blue or purple) Gram (Red)

Depends on Gram staining Technique.

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Gram Staining Procedure

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Gram Staining Procedure

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Gram Positive Cell Wall:

Several layers of peptidoglycan Teichoic acids unique to Gram-positive cell wall Gram Positive organisms appear purple Sensitive to penicillin and lysosome 50% peptidoglycan layer
20-80 nm thick

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Gram-positive cell wall

Gram Negative Cell Wall:

Outer membrane Thin peptidoglycan layer 10-20nm thick Periplasmic space

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Gram Negative Cell Wall

Outer membrane:
Unique in Gram Negative cell walls Bilayer outside of Peptidoglycan, intercalated with proteins Permeability barrier lipopolysaccharide LPS ( endotoxin ) Porins Lipoprotein
anchors to cell memb

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Lipid Polysaccharide LPS:

The LPS molecule - hydrophobic region, called Lipid A, Core polysaccharide - Hydrophilic O-specific polysaccharide Lipid A endotoxin Activates compliment Activates clotting factors Activates macrophages Leads to endotoxin shock O-specific polysaccharide ( O antigen ) Adhesion Inhibits phagocytosis Antigenic
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Gram negative peptidoglycan:

Thin 10-20 nm

Periplasmic space
Enzymes For Assembly Of Cell Wall And Membrane Components, Various Degradative Or Detoxifying Enzymes, Secretion Systems, Sensing Proteins For Chemotaxis Signal Transduction. Endo flagella in periplasm in spirochetes
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Cell Membrane
Similar to eukaryotes Permeability barrier No sterols and may have saturated fatty acids Functions:
1. Osmotic or permeability barrier 2. Location of transport systems 3. Energy generating functions, involving respiratory and photosynthetic electron transport systems, establishment of proton motive force, and ATP-synthesizing ATPase 4. Synthesis of membrane lipids (including lipopolysaccharide in Gram-negative cells) 5. Synthesis of murein (cell wall peptidoglycan) 6. Assembly and secretion of extra cytoplasmic proteins 7. Coordination of DNA replication and segregation with septum formation and cell division 8. Chemo taxis 9. Location of specialized enzyme system

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Mesosomes : Invagination of cell membrane Energy production electron transport chain DNA replication and segregation, cell wall synthesis, or increased enzymatic activity Plasmids : Extra chromosomal DNA molecule replicate independently
F plasmid Fertility R Plasmid Resistance Col plasmid Bacteriocins Ti plasmid virulence
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Ribosomes:
70s ribosomes

Smaller than Eukaryotic ribosomes

70S ribosomes in eucaryotic mitochondria and chloroplasts contain

ssrRNA closely related to bacterial ribosomal RNA.

This is taken as a major line of evidence that these organelles are

descended from procaryotes.

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DNA Nucleoid
Typically One Large Circular Molecule Of DNA Coiled And Supercoiled And Anchored By Proteins Meiosis And Mitosis Are Absent Replication By Binary Fission

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Inclusions
Usually Reserve Materials Glycogen Inclusions In E.Coli For Energy Source. Polyphosphate (Volutin Granules) In Corynebacteria For Energy Source

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Endospores:
Formed intracellularlly in unfavorable conditions Resist to desiccation, radiation, heat, antiseptics

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Endospores are produced as intracellular structures within the cytoplasm of certain bacteria, most notably Bacillus and Clostridium species.

Endospore forming bacteria left to right: Clostridium botulinum, Bacillus brevis, Bacillus thuringiensis

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Endospore formation is NOT a mechanism of reproduction. Rather it is a mechanism for survival in deleterious environments. During the process of spore formation, one vegetative cell develops into one endospore.

The sequential steps of endospore formation in a Bacillus species. The process of endospore formation takes about six hours. Eventually the mature endospore is released from its mother cell as a free spore Free endospore

Endospore within mother cell

Vegetative cell

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Under favorable nutritional and environmental conditions, an endospore germinates into a vegetative cell.

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Classification of Bacteria

Shape Gram staining Respiration

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Shape
Cocci Bacillus Coccobacillus

Gram Stain
Gram positive Bacteria - Blue Gram negative Bacteria RED

Spiral
Diplococcus

Pleomorphic

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Respiration
1. Obligate aerobes ex: Mycobacterium

2. Facultative Anaerobes ex: most bacteria

3. Microaerophilic ex: Campylobacter

4. Obligate anaerobes ex: Clostridium

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