GENERAL MICROBIOLOGY

OUTCOMES OF GENERAL MICROBIOLOGY

By the end of the sessions, learners will be able to:

Differentiate among the major characteristics of each group of microorganisms. Identify the basic shapes of bacteria Describe the structure and organization of bacteria Compare and contrast the cell walls of gram-positive & gram negative bacteria Compare and contrast prokaryotic and eukaryotic cells Describe Bacterial requirement for growth Classify bacteria on the basis of preferred temperature range Explain how microbes are classified on the basis of oxygen requirement Describe Bacterial growth curve Distinguish between different types of culture media Identify bacterial viruses (bacteriophages) Discuss bacteriophages propagation (replication) Describe the mode of actions of antibacterial chemotherapeutic agents Discuss antibiotic susceptibility testing Identify the complications of chemotherapy Describe the mechanisms of bacterial resistance to antimicrobial agents Define Sterilization, Disinfection and antisepsis Describe principles and applications of sterilization and disinfection Define infection, specify the stages of the infectious process Explain microbial virulence

Recommended books, periodicals and web sites

Jawetz, Melnick, & Adelberg's Medical Microbiology Lang Medical Books/ McGraw-Hill (review of medical microbiology)Greenwood et al. Medical MicrobiologyWarren Livenson, Review of Medical microbiology & immunology

1. www.asm.org 2. www.virolgy.net/ 3.www.pathmicro.med.sc.edu/book/wlcome.htm4. www.youbundle.com/.../immunolgy-goldsby-immunology5.http://www.biology.arizona.edu/immunology/microbiology_immunology.html6. http://www.med.sc.edu:85/book/welcome.htm7. http://www.mic.ki.se/Diseases/c2.html8.http://www.tulane.edu/~dmsander/Big_Virology/BVHomePage.html9. http://www.hepnet.com/hepb.htm10. http://whyfiles.org/012mad_cow/7.html 11.http://www.microbelibrary.org/12. asmnews@asmusa.org13.http://www.phage.org/black09.htm14.http://www.microbe.org/microbes/virus_or_bacterium.asp 15.http://www.bact.wisc.edu/Bact330/330Lecturettopics

OUT COMES of Bacterial Structure

By the end of the sessions, learners will be able to:

Differentiate among the major characteristics of each group of microorganisms. Identify the basic shapes of bacteria Describe the structure and organization of bacteria Compare and contrast the cell walls of Gram-positive & Gram negative bacteria Compare and contrast prokaryotic and eukaryotic cells

Classification of Microorganisms

Microorganisms are either :

Prokaryotes e.g. bacteria or Eukaryotes e.g. fungi

EUOKARYOTESeuo=true, karyote=nucleusAlgae, protozoa, fungi.Cells of higher plants and animalsTrue nucleus, organelles, mitochondria,Ribosomes (protein + RNA): 8O S (sedimentation constant) Divide by mitosis (& sexually)

PROKARYOTESwithout true nucleusBacteria, mycoplasma, rickettsia, chlamydiaHereditary material suspended in a portion of the cytoplasm (not true nucleus) No nuclear membraneNo organelles Ribosomes: 70 SNo mitochonderiaDivide by simple binary fission

BACTERIA: THEIR STRUCTURES & ORGANIZATION

Bacterial MorphologyMorphological features: shape, size, arrangement, staining characteristics.

Bacterial size: 0.2-1.2 m width, 0.4-14 m length

Bacterial shape & arrangement: (A) cocci (coccus): diplo, clusters, chains. (B) bacilli (bacillus): single, in pairs or chains. (C) Spirals: rigid or flexible.

Staining characteristics:

2. Bacilli or cylindrical:

3. Spiral:

VibrioSpirochetes

Staining characteristics: kinds of stains: Simple, Differential. Most important differential stain in clinical microbiology is:Grams stain: it divides bacteria into Gram+ve bacteria (violet staining) & Gram ve bacteria (red staining). The 2nd important stain is Ziehl-Neelsen stain, used to identify genus Mycobacteria.

BACTERIAL ULTRA-STRUCTURE &

THEIR FUNCTIONS

Prokaryotic cell

Bacterial Structures

Bacterial CoatsCytoplasmic structuresExternal Appendages CapsuleNucleoid materialFlagellaCell wallRibosomePili (Fimberia)Cell membraneInclusion granules

Mesosome

Cytoplasm * It contains :

- Nucleoid : Bacterial chromosome (1 mm long) single circular double stranded DNA

- Plasmids : circular double stranded extrachromosomal DNA molecules

Ribosomes : site of protein synthesis ( 70S )

Inclusion granules : phosphates,sulpher,lipids,CHO---energy reserve, ATP synthesis

Mesosomes : cell division, sporulation, respiratory enzymes

Cytoplasmic structures:

- Single chromosome formed of single circular molecule of ds DNA. - Carries the genetic characters of the bacterium.N.B.: Additional extrachromosomal Genetic materials may be present in the bacterium see later. 1- Nucleoid

2- Ribosomes: site of protein synthesis ( 70S )

3- Inclusion granules: - These are granules of food & \ or energy reserves. - Phosphate (used for synthesis of ATP) is stored in metachromatic or volutin granules.

4- Mesosomes:

- Composition: inward invaginated convoluted tubules of the cytoplasmic membrane into the cytoplasm.

- Functions:Involved in the mechanisms of cell division and sporulation. Respiratory function: providing a membranous support for respiratory enzymes.

Bacterial Coats1- Cell membrane: - Composition: Bilayer of phospholipoprotein (similar to that of eukaryotic cells but without sterols)

Functions of cell membrane:

1. Selective transport: simple diffusion, facilitated diffusion and active transport. 2. Excretion of extracellular enzymes: a Hydrolytic enzymes: which digest large food molecules b Destructive enzymes: used to destroy harmful chemicals e.g. antibiotics. 3. Respiration: Due to presence of respiratory enzymes 4. Reproduction. 5. Cell wall biosynthesis. 6. Chemotactic system

2- Cell wall:

- Composition:- The cell wall of bacteria is a complex structure.

Its strength is due to peptidoglycan [PG] (a complex polymer consisting of Nacetylglucosamine [NAG] and Nacetylmuramic acid [NAM]

- Besides PG, additional components in the cell wall divide bacteria into Grampositive and Gramnegative.

Grampositive cell wall is composed of:

Peptidoglycan: There are as many as 40 sheets of PG, comprising up to 50% of the cell wall material. Teichoic acids: - They are the polymer of ribitol or glycerol phosphate. - Teichoic acids and cell wall associated proteins are the major surface antigens of the Grampositive bacteria.

Gramnegative cell wall is composed of:

PG: It is much thinner, composed of only 1-2 sheets comprising 510% of the cell wall material.Outer membrane: - It is phospholipid protein bilayer present external to the PG. - The outer surface of the lipid bilayer is composed of molecules of Iipopolysaccharide (LPS) which consists of a complex lipid called lipid A (endotoxin) chemically linked to polysaccharides which represent the major surface antigen of Gm-ve bacteria (somatic O antigen).Periplasmic space: It is the space between the cytoplasmic and outer membranes. It contains the PG layer and a gellike solution of proteins.

Cell Wall: Gram-Negative & Gram-Positive

Functions of the cell wall

1. It maintains the characteristic shape of the bacterium. 2. It supports the weak cytoplasmic membrane against the high internal osmotic pressure. 3. It plays an important role in cell division. 4. It is responsible for the staining affinity of the organism.5. It protects the organism from external environmental hazards.

Wall deficient variants(Bacteria without cell wall)

MycoplasmaL FormsNaturally presentArtificially induced due to treatment of the cell wall by antibiotics or lysosymsCan replicate Cant replicateCant reassume a walled formMay (or may not) reassume the walled form upon removal of the causative agents. - Have no defined recognizable shape. - Not stained by gram stain. - Resistant to wall inhibitor antibiotics.

3- Capsule and Related Structures:

- Composition: The outer most layer in some bacteria that is formed only inside the host (In vivo) It is formed of polysaccharide (except B. anthracis capsule formed of polypeptide).

Functions: A- Protection: * Against phagocytosis (virulence factor). * Against various kinds of antibacterial agents, e.g. bacteriophages, colicins, complement and lysozymes. B- Attachment to host surface: By capsules or glycocalyx in order to establish infection, e.g. Streptococcus mutans form glycocalyx to stick to the tooth enamel.

Capsulated bacteria

Variants of the capsule: 1- Capsule: - A well defined layer that adhere to the bacterium surface all-around. - It may be in the form of large capsule, small capsule or microcapsule (electron dense layer). 2- Slime layer: It is a surface layer that is loosely distributed all-around the bacterium. 3- Glycocalyx: It is a loose meshwork of polysaccharide fibrils extending outwards from one pole of the bacterium. N.B.: All capsule variants are not stained by Gram stain and appear as hallow zones around the organism.

External appendages: 1- Flagellum:

- Composition: long tubular external appendages which is formed of protein called flagellin (antigenic protein and form the H antigen of bacteria)

- Function: flagellum is the organ of motility of the bacteria [motility chemotaxis may be toward (+ve) or away (-ve) from certain area]

A. Monotrichous: One flagellum

B. Lophotrichous: multiple polar flagella C. Amphitrichous: a single flagellum on each of two opposite ends D. Peritrichous: Flagellae around the cell Flagella arrangement Location and number of flagella

E.M. picture of flagellated bacilli

2- Pili or fimbriae:

- Composition: Thin short tubular external appendages which are formed of protein called pillin (non antigenic protein).

- Types and Functions: A- long pilus: also called the sex pilus (F or fertile pilus) is involved in the transfer of DNA between bacteria, a process known as conjugation. B- Short pilus: also called fimberia and it adheres the organism to the host surface (a virulence factor).

Pili (fimbriae)

Comparison between flagella and pili

FlagellaPiliMorphologyTall, thin & tubularShort, thin & tubularCompositionFlagellin proteinPillin proteinAntigenicityAntigenicNot antigenicFunctionsMotility of the organismAttachment to the host- Gene transfer by conjugation

Bacterial Spores(Endo-spores)Definition: a highly resistant resting form (phase) of some bacteria (genera Bacillus and Clostridium) that is formed on exposure to bad external environmental conditions.

- Sporulation: it is a process by which a single vegetative bacterium forms a single spore on exposure to bad external environmental conditions ( in vitro).

N.B.:Endospore does not grow or reproduce, and exhibits absolute dormancy.

Mechanism of sporulation: - The cell membrane invaginates enclosing a section of the cytoplasm that contains the bacterial chromosome, some ribosomes and other cytoplasmic materials that will be needed for germination. - It acquires a thick cortex and a thin but tough outer spore coat.

Germination: It is the process by which the endospores can respond quickly to changes in the environment returning to the vegetative state within 15 min.

- Mechanism of germination: In the process of germination, the spores absorb water and swell, the protective coat disintegrates and a single vegetative cell emerges.

Viability and resistance of the spore: - The spores are highly resistant to disinfectants, drying and heating and only moist heat at 121 C for 10 20 minutes is able to kill spores. - The marked resistance of the spores is due to: 1- High content of Ca 2+ and dipicolinic acid 2- The impermeability of their cortex and outer coat. 3- Their low content of water. 4- Their very low metabolic and enzymatic activity.

Morphological characters of endospores: 1. Staining: Using Gram's stain, the spore remains uncoloured and can be seen as a clear area within the stained cell. The spores can be stained using special procedures.2. The position: In relation to the body of the bacillus, the spore may be central, terminal or subterminal.3. The shape: The spores may be oval or rounded.N.B.: The position and shape of spores are characteristic of the species and may help in the microscopic identification of the bacterium.

Characteristics of SporesMorphologyStainingPosition

Comparison between bacterial capsule and bacterial endo spore

CapsuleSporeSite of formationIn vivo (inside the host)In vitro (in the environmentMetabolic activity and replicationThe organism is active and can replicateThe organism is inactive and cant replicateFunctions Adhesion to the host, protection and antiphagocyticProtection from bad conditions of the environment

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