Prokaryotic Cell

Cytoplasmic structures
a. No membrane bound structures
b. DNA consist of singular circular chromosomes
c. Attach to a mesosome which is a saclike structure
Bacterial Ribosomes are found free to attached to the cytoplasmic
granules
Consist of polysaccharide such a poly - typing
1. Cell Envelope structure
I. Cell Membrane
a) Lipoprotein membrane that surrounds the cell wall
b) Location of the electron transport Chain
c) Made of phospholipids and proteins but no sterols
Functions:
Regulates transport across membrane acts as osmotic
barrier
2. Cell Wall
a) Rigid structures that maintains the shape of the cell
b) Prevents bursting of the cell (cause by high osmotic
pressure)
Gram Positive Cell Wall – Peptidoglycan (antibiotics)
Glycan Chains- NAG and NAM
Techoic Acid(peptidoglycan); lipotechoic acid (Plasma
membrane)
Gram Negative Cell Wall- inner peptidoglycan (thinner than
gram positive cell walls)
Outer Membranes
Proteins, lipids and LPS
LPS -antigenic O-specific polysaccharide
-core polysaccharide
-inner lipid A (endotoxin) –fever and shock
conditions

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

I. Periplasmic Space
Gel- like matrix
Nutrient binding proteins
Degradative and detoxifying enzymes
II. Acid Fast Cell Wall
Waxy layer and fatty acid (Mycolic Acid- mycobacterium spp.)
 ABSENCE OF CELL WALL
Mycoplasma 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 wall

a. Gr (+) turns to a Protoplast
b. Gr (-) turns to a Spheroplast- Major portion of the cell
wall will remain

3. Surface Polymer
Capsules- discrete organized covering Polysaccharide, polypeptides;
acts as virulence factors to evading phagocytes
Slime 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 Appendages
FLAGELLUM- organ of locomotion

BACTERIAL SPECIES VARY IN THEIR POSSESION OF FLAGELLA

None - atrichous
Extend to one end - polar

AXIAL FILAMENTS
Singly - monotrichous
At one or both ends - amphitrichous
Multiply in tuft at one - Lophotrichous
Occurs on sides of the bacterium -peritrichous

PILI (FIMBRIAE)
Hair-like protein structures
Aid in attachment to surfaces
a. Sex pili-bacterial conjugation and gene exchange
b. Adherence pili- allowing them to attach to specific
eukaryotic host cells

STAINING
Unstained/Unfixed Preparation
Wet Mount
Hanging Drop
-Natural conditions suspended in fluids
-Bright Field, Dark Field and Phase Contrast Microscope
-Motility
Brownian Movement- along with the fluid
True motility- with locomotor
-Morphology
Dye (Salt)
Chromophore
Color of the basic dye- Positive ion
Color of the Acidic dye-Negative ion
Bacteria are slightly negatively charged at pH 7
The colored positive in a basic dye is attracted to the negatively
charged bacterial cells.
Fixed/Stained Preparation

Smear---- Heat Fixed---------- Cell’s molecules change shape

Chemical Fixation--------No destruction of structures
Staining
Increase visibility
Reveal additional information about the bacteria
TYPES OF STAINING
A. Simple Staining
-single dye
-cells and structures stain the same color
Characteristic 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 structures

C. Gram Staining
Gram Positive and Gram negative
Most important staining
Determine the source of microbes as contaminants
Fundamental Step in the diagnosis and treatment of diseases
Determine the most effective antibiotics for critical ill patients

THEORIES
 1. Membrane Theory of Knaysi
Gram 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 Theory

The 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 STAIN

Identification 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 STAINS
Selective Stain specific structures
Endospores
Capsules
Flagella
Granules
Cell wall
Nucleus

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 Negative

Gram Reaction Purple Red
Peptidoglycan layer Thick Thin
Techoic acid Present Absent
LPS content Absent Present
Periplasmic Space Absent Present
Outer Membrane Absent Present
Lipid content and Low High
lipoprotein
Resistance to Physical High Low
Disruption
Lyzosyme Cell wall High Low
disruption
Susceptibility to High Low
penicillin
Susceptibilty to Low High
streptomycin,
chlorampenicol and
tetracycline
Inhibition by basic High Low
dyes
Susceptibility to High Low
Anionic detergents
Resistance to Sodium High Low
Azide

Steps in Gram Staining

Steps Reagent Remarks

Primary or initial Crystal Violet Colors cytoplasm purple
Stains regardless of cell type
Mordant Iodine Combines with crystal
violet to form insoluble
complex
Decolorizer Alcohol or Acetone Purple dye is retained
Alcohol by gram positive cells
but is readily removed
from gram negative
cells
Secondary or Counter Safranin Stains the colorless
Stain gram negative cell to
red while Gram positive
cells remain purple
Gram Positive- Purple
 Gram Negative- Red

Steps in Acid Fast Staining

Steps Reagent Remarks

Primary or initial Stains Carbolfuchsin All cells are color Red
Mordant Fixes the stain and
Physical Steam Heat facilitates entrance of
Chemical Phenol the dye

Decolorizer Acid- Alcohol Non-Acid Fast are

decolorized; acid fast
remain color red
Secondary or Counter Methylene Blue Stains the colorless and
Stain non acid fast blue while
acid fast remains
colored red
Non- Acid fast bacilli- Blue
Acid fast bacilli- Red