BACTERIAL CELL ULTRASTRUCTURES

General Division:
I. External - Capsule - Fimbriae - Flagellum Cell Wall Cytoplasmic Membrane Cytoplasm Mesosome Nuclear Body Ribosome Plasmid Inclusion Granule Endospore

II. Internal

BACTERIAL ULTRASTRUCTURE A. Bacterial Surface Coating: = are extracellular polysaccharide polymers which surrounds the bacterial cell Capsule = well-formed thick viscous jelly-like structure firmly attached to the cell wall surrounding the cell = not readily removed. = easily visualized by negative staining using India ink method

Slime layer = loose and irregularly arranged meshwork of fibrils totally detached from the cell but still surrounds the cell = when present are more easily washed off

Chemical Comp.: = water main component 98-99% = 1-2% of bacterial capsule are chemically polysaccharide, Except capsule of: B. anthracis-polypeptide (D-glutamic acid) S. pyogenes-Hyaluronic acid Function: = protection from phagocytosis = correlates with virulence = act as antigen (used in the identification) and typing of capsulated bacteria = allows bacteria to adhere/attach to various surfaces in its natural environment in order to survive Detection: 1. Negative staining (India Ink) 2. Special staining (Capsular method) 3. Serological (Quellung reaction)

B. Appendages

Flagellum & Axial filaments Fimbriae

FLAGELLUM: = long, thick, helical protein filament of uniform length and diameter = commonly seen among free-swimming bacteria = originates in cytoplasmic membrane = composed of 3 parts: 1. Basal body anchors the flagellum to the cell wall and plasma membrane 2. Hook attached to the basal body and connects to the filament external to the cell and contains the flagellar protein

3. Filament

Chemical Comp.: protein monomer - Flagellin Function - Motility organelle of many pathogenic bacteria Detection: 1. Darkfield / Phase contrast microscopy (Wet mount / Unstained smear) 2. Brightfield/Electron Microscopy using stained specimen 3. Serological use of specific antisera against flagellar (H) antigen 4. Swarming phenomenon 5. Motility medium 6. Hanging drop preparation

Types and Arrangement of flagella in relation to the distribution and number:

I. Monotrichous

single polar flagellum at one end

II. Multitrichous

more than one flagella

Types: A) Lophotrichous

tuft of flagella at one polar end

B) Amphitrichous

single flagellum or tulf of flagella at both polar end

C) Peritrichous

flagella distributed all over the body of the bacteria

Axial Filaments = flagella-like sheathed filaments located in the periplasmic space (between the inner) and outer membrane of the cell = move by traveling helical wave on opposite direction = motility organelle of spirochete

Fimbriae / Pili / Microfibrils = short, straight, thin hair-like filaments usually distributed around the body of bacteria. = originates in the cytoplasmic membrane = found virtually among all gram (-) bacteria but not in gram (+) bacteria

2 types according to function: 1. Common/ordinary for attachment or adherence to mucosal surfaces of host cell during colonization and infection. 2. Sex pili responsible for attachment of donor and recipient cell during bacterial conjugation whereby genetic material (DNA) from one cell is transferred to another (reproduction). Chem. Comp. - Protein (Pilin) Detection: Electron Microscopy

Cell wall (Peptidoglycan, Murien, Mucopeptide / Glycopeptide) = complex, rigid, multilayered structure that protects the protoplast and the underlying fragile plasma membrane = found in all pathogenic free-living bacteria except Mycoplasma (cell-walless bacteria) Chemical Comp.: 1. Protein (Mesodiaminopimelic acid,Isomers of D-glutamic) acid and D-alanine 2. Polysaccharide (N-acetylmuramic acid and N-acetylglucosamine) = responsible for rigidity of cell wall 3. Lipids

Function : 1. Responsible for the characteristic shape of bacterium 2. Provides strong structural support necessary to keep bacterial cell from rupturing due to changes in the environmental osmotic pressure 3. Contain somatic O antigen that can serologically identify particular bacteria 4. Site of action of some antibiotics 5. Determines differences in gram staining reaction Detection : 1. Microscopy of smear prepared from special staining method. 2. Electron microscopy. 3. By chemical methods using lysozyme.

COMPARISON OF GRAM POSITIVE AND GRAM NEGATIVE CELL WALL

Gram Positive Peptidoglycan
Very

Gram Negative complex layer composed of thin peptidoglycan with few crossedcrossed-linked and an outer membrane layer. Constitute 5-10% of the cell 5wall material. teichoic acid Outer membrane contains: PhospholipidsPhospholipids-located in the inner membrane Lipoprotien connects OM to the peptidoglycan Lipopolysaccharide (LPS/Endotoxin layer) located in the outer layer of the OM - contains the lipid A
Resistant Resistant No More

thick homogenous single layer extensively crossed-linked. crossedConstitute 50-90% of the cell wall 50material. (NAM, NAG, D-alanine) DDiaminophemilic acid

Auxiliary compounds

acid (Polymer of ribitol and) glycerol phosphate Lipoteichoic acid Surface protein
Teichoic

Penicillin sensitivity Response to lysozyme

Sensitive Digest/

Degrade

Gram Positive

Gram Negative

Damage to the cell wall may result to: = when a gram positive bacteria is exposed to lysosyme will degrade the peptidoglycan layer resulting to complete removal of the cell wall producing a osmotically fragile wall-less spherical body called Protoplast = since all cell wall components is removed, therefore incapable of regeneration Spheroplast = when a gram negative bacteria is exposed to lysozyme it looses the peptidoglycan layer but retain the outer membrane leaving a less fragile spherical body capable of regenerating the cell wall.

Protoplast: comprises the naked cytoplasmic membrane and its content PERIPLASM = space between the plasma inner membrane and the outer membrane layer = readily observe among gram negative bacteria = consist of a gell-like substances which help secure nutrients from the environment CYTOPLASMIC/PLASMA/CELL MEMBRANE: = thin elastic bilayered semi-permeable membrane lying underneath the cell wall enclosing the cytoplasm of the cell. Chemical Composition - Lipoprotein (composed of phospholipids and protein molecules)

Functions: 1. Serve as selective permeability barrier (transport of nutrient material in & out of cell) 2. Site of electron transport and oxidative phosphorylation of substances involve in the generation of chemical energy (ATP) 3. Helps in the excretion of metabolic waste product 4. Site for excretion of enzyme involved in OM synthesis, CW synthesis and in the secretion of extracytoplasmic & extracellular substances: A. Permease for active uptake of nutrient B. Respiratory C. Polymerase that manufacture substances of the cell wall D. Hydrolytic Detection - Electron microscopy

Cytoplasm = refers to everything that is enclosed by the cytoplasmic membrane = site for most bacterial metabolism = 80% is composed of water MESOSOME: = usually seen as an invagination of the membrane associated cytoplasmic sac seen in Gram (+) cell = contains lamellar, tubular and vesicular structure = often associated with division septa Chemical Composition - Lipoprotein Functions: 1. Site for the synthesis of cytochrome oxidase and reductase enzyme 2. Provide support for enzymes present in the cell membrane 3. Responsible for compartmenting DNA during cell division and sporulation Detection- Electron microscopy

NUCLEAR BODY: (Nucleus / Nucleoid) = genetic material of bacteria = contains a single circular molecule of double stranded DNA network which runs parallel to the long axis of the cell = found homogenous within the cytoplasm not enclosed in a nuclear membrane. = constitute 2-3% of the cell weight. = no definite form and seen at all stages of growth cycle of the bacteria. Chemical Composition DNA Function: 1. Controls the growth and metabolic activity of the bacteria 2. Responsible for hereditary characteristics of the cell. Detection- Electron microscopy, Feulgin staining (+)

PLASMID/EPISOME: = extrachromosomal genetic material capable of autonomous replication usually located near the chromatin body. Chemical Composition DNA

Function: 1. Transfer of genetic material from one cell to another by conjugation (Transmissible Plasmid) 2. Carry genes for activities like: a) Antibiotic resistance b) Toxin production c) Synthesis of enzyme d) Tolerance to toxic metals Detection Feulgin staining (+), Electron microscopy

RIBOSOME: = histone-like particles composed of ribosomal RNA (rRNA) and protein molecules found in the cytoplasm of the cell. = site of action for many antibiotics that inhibit protein synthesis. = have a sedimentation coefficient of 70S and are composed of 50 S and 30 S subunits containing 16 S, 23 S and 5 S RNA respectively = procaryotic ribosome - 70s = eucaryotic ribosome - 80s Function Site of protein synthesis

Detection Feulgin staining (-), Electron microscopy

INCLUSION / CYTOPLASMIC GRANULE: = large granules found anywhere in the cytoplasm = are accumulation of organic and inorganic substances which serves as source of energy and nutrient supply to the organism Kinds: 1. Babes Ernst / Volutin / Metachromatic granules Chemical Composition polymerized metaphosphate which can be used in the synthesis of ATP Function reserve energy supply

Demonstration simple staining / special staining (Albert s stain)

2. Lipid granules Chemical Composition Function polymer of betahydroxybutyric acid reserve food supply.

3. Sulfur granules derived energy by oxidizing sulfur and sulfur-containing compounds. Function reserve energy supply for sulfurcontaining bacteria. polymer of glucose.

4. Glycogen granule Function

reserve food supply.

ENDOSPORES: = highly refractile body formed within vegetative bacterial cells in response to adverse environmental condition and due to deficient nutrient supply = known as endospore, because it is formed inside the bacteria cell and is genetically controlled = are metabolically inactive bacterial cell that are highly resistant to desiccation, heat and various chemical agents = found in the cytoplasm of rod-shaped sporeforming bacteria of the genus Bacillus & Clostridium

= Composed of five parts: 1. Core located at the center which is the spore cytoplasm/protoplast. - contains the nucleus and enzyme dipicolinic acid 2. Spore wall enclosing the core. 3. Cortex laminated structure surrounding the spore wall 4. Spore coat multilayered membrane enclosing the cortex 5. Exosporium the outermost covering of the spores which give the spore a rigid appearance.

= contains Calcium Dipicolinate which aids in heat resistance within the core by making the protein and nucleic acid more resistant to denaturation = germinate under favorable nutritional condition after an activation process that involves damage to the spore coat = helpful in identifying some specie of bacteria especially those sporeforming bacilli (Clostridium and Bacillus)

Classification accdg. to: A. Location - Central, Subterminal, Terminal

B. Shape - a) Ovoid b) Circular C. Swollen/not swollen (swollen when the diameter is more than the of the) bacterial cell, not swollen diameter the same as bacterial cell

Detection: 1. Microscopy of stained smear A) Negative staining B) Positive staining 2. Phase contrast microscopy