Prokaryotic Cell Cytoplasmic structures

a. No membrane bound structuresb. DNA consist of singular circular chromosomesc. Attach to a mesosome which is a saclike structureBacterial Ribosomes are found free to attached to the cytoplasmic granulesConsist of polysaccharide such a poly - typing

1. Cell Envelope structure I. Cell Membrane

a) Lipoprotein membrane that surrounds the cell wallb) Location of the electron transport Chainc) Made of phospholipids and proteins but no sterols

Functions:Regulates transport across membrane acts as osmotic barrier

2. Cell Walla) Rigid structures that maintains the shape of the cellb) Prevents bursting of the cell (cause by high osmotic

pressure)Gram Positive Cell Wall – Peptidoglycan (antibiotics)

Glycan Chains- NAG and NAMTechoic Acid(peptidoglycan); lipotechoic acid (Plasma

membrane)Gram Negative Cell Wall- inner peptidoglycan (thinner than gram positive cell walls)

Outer MembranesProteins, lipids and LPSLPS -antigenic O-specific polysaccharide

-core polysaccharide-inner lipid A (endotoxin) –fever and shock

conditions

Functions:a. Barrier to hydrophobic compoundsb. Acts a sieve (through porins)c. Attachment sites

I. Periplasmic SpaceGel- like matrixNutrient binding proteinsDegradative and detoxifying enzymes

II. Acid Fast Cell Wall Waxy layer and fatty acid (Mycolic Acid- mycobacterium spp.)

ABSENCE OF CELL WALLMycoplasma and Ureaplasma

- Lacks Cell wall but they have sterols

L-Forms -Media supplemented with sugar or serum to prevent

osmotic rupture- capable of reverting to its normal form (residual

peptidoglycan)

Using Physical and Chemical Agents to lose Cell walla. Gr (+) turns to a Protoplastb. Gr (-) turns to a Spheroplast- Major portion of the cell

wall will remain

3. Surface PolymerCapsules- discrete organized covering Polysaccharide, polypeptides;

acts as virulence factors to evading phagocytesSlime layer- more diffuse polysaccharide inhibits phagocytosis,

aid in the adherence to host tissue or synthetic implants.Glycocalyx- tangled mass of thin polysacharride fibers

4. Cell AppendagesFLAGELLUM- organ of locomotion

BACTERIAL SPECIES VARY IN THEIR POSSESION OF FLAGELLANone - atrichousExtend to one end - polar

AXIAL FILAMENTSSingly - monotrichousAt one or both ends - amphitrichousMultiply in tuft at one - LophotrichousOccurs on sides of the bacterium -peritrichous

PILI (FIMBRIAE)Hair-like protein structuresAid in attachment to surfacesa. Sex pili-bacterial conjugation and gene exchangeb. Adherence pili- allowing them to attach to specific

eukaryotic host cells

STAINING

Unstained/Unfixed PreparationWet MountHanging Drop

-Natural conditions suspended in fluids-Bright Field, Dark Field and Phase Contrast Microscope

-MotilityBrownian Movement- along with the fluidTrue motility- with locomotor

-MorphologyDye (Salt)

ChromophoreColor of the basic dye- Positive ionColor of the Acidic dye-Negative ion

Bacteria are slightly negatively charged at pH 7The colored positive in a basic dye is attracted to the negatively

charged bacterial cells.Fixed/Stained Preparation

Smear---- Heat Fixed---------- Cell’s molecules change shapeChemical Fixation--------No destruction of structures

StainingIncrease visibilityReveal additional information about the bacteria

TYPES OF STAININGA. Simple Staining

-single dye -cells and structures stain the same colorCharacteristic of size, shape and cell arrangement

B. Differential Staining-distinguish structures within the cell, or by different types of cells- By staining them with different colors

React with specific microbial structuresC. Gram Staining

Gram Positive and Gram negativeMost important stainingDetermine the source of microbes as contaminantsFundamental Step in the diagnosis and treatment of diseasesDetermine the most effective antibiotics for critical ill patients

THEORIES

1. Membrane Theory of KnaysiGram negative bacteria have a high lipid content; alcohol added removes the

lipid the gram negative cells, thereby increasing cell permeability resulting in the loss of the dye complex

2. Isoelectric Theory

Gram positive have lower isoelectric point or the pH at which the cells have no change. This makes the cells more susceptible to the entrance of the dye and slow to decolorization

3. Outer Surface TheoryThe out surface of the cell wall of Gram Positive bacteria contains Mg+RNA

which forms a complex crystal violet iodine. This complex is insoluble with alcohol

D. ACID FAST STAINIdentification of genus Mycobacterium

Mycobacterium contains high levels of lipid material (mycolic acid) that repel water-soluble dyes that are difficult to stain by standard procedures

All mycobacteria are acid fast

E. STRUCTURAL STAINSSelective Stain specific structures

EndosporesCapsulesFlagellaGranulesCell wallNucleus

Culture Media Right nutrients in their concentration Properly adjusted pH Sufficient Moisture Desired consistency Free inhibitory substances Initially sterile

Comparison of Gram Negative from Gram Positive

Gram Positive Gram NegativeGram Reaction Purple Red

Peptidoglycan layer Thick ThinTechoic acid Present AbsentLPS content Absent Present

Periplasmic Space Absent Present

Outer Membrane Absent PresentLipid content and

lipoproteinLow High

Resistance to Physical Disruption

High Low

Lyzosyme Cell wall disruption

High Low

Susceptibility to penicillin

High Low

Susceptibilty to streptomycin,

chlorampenicol and tetracycline

Low High

Inhibition by basic dyes

High Low

Susceptibility to Anionic detergents

High Low

Resistance to Sodium Azide

High Low

Steps in Gram Staining

Steps Reagent RemarksPrimary or initial

StainsCrystal Violet Colors cytoplasm purple

regardless of cell typeMordant Iodine Combines with crystal

violet to form insoluble complex

Decolorizer Alcohol or Acetone Alcohol

Purple dye is retained by gram positive cells but is readily removed

from gram negative cells

Secondary or Counter Stain

Safranin Stains the colorless gram negative cell to

red while Gram positive cells remain purple

Gram Positive- PurpleGram Negative- Red

Steps in Acid Fast Staining

Steps Reagent RemarksPrimary or initial Stains Carbolfuchsin All cells are color Red

MordantPhysicalChemical

Steam HeatPhenol

Fixes the stain and facilitates entrance of

the dye

Decolorizer Acid- Alcohol Non-Acid Fast are decolorized; acid fast

remain color redSecondary or Counter

StainMethylene Blue Stains the colorless and

non acid fast blue while acid fast remains

colored redNon- Acid fast bacilli- Blue

Acid fast bacilli- Red