CELL WALL

allows us to classify gram+ & gram-

structure found beneath the capsule (for capsulated microorganisms)
major structural component of the bacterial cell, makes up the shape
rigid, relatively not elastic, highly ductile, high tensile strength
10-20% dry weight of the cell; 10-20 nanometer in thickness
contains spores (1 nanometer in diameter)

peptidoglycan - reason for high tensile strength

-murein
-mucopeptide

FXNS:

maintains the morphology of the cell; gives rigidity and shape to the cell
protects from osmotic lysis
site of various antigenic determinants of the cell surface

o + Teichoic acid
o - polysaccharides
provides the support necessary for propulsion by flagella
site of synthesis of macromolecules
provides protection from some antibiotics and destructive chemicals
determines differences in grown stain reaction

2 CLASSES:
1. GRAM POSITIVE
peptidoglycan - outermost part
amino acid + sugar
single giant molecules consists of 40 sheets
comprising 90% of cell wall
attacked by the antimicrobial agent

N-Acetylglucosamine [insert picture]

2 Specialized components:
a. Teichoic Acid - H2O soluble polymer containing glycerol residues joined through
phosphodiether linkages
- in Streptococcus pneumonae it is called Forrsman antigen

FXNS:

major surface antige of gram+ bacteria

 binds Mg2+ ion that plays a role in the supply of this ion to the cell

o Mg2+ stabilizes the cell membrane
involved in the activation of autolytic enzymes and in determining the susceptibility of
the wall to autolytic activities

o autolytic enzymes causes lysis on itself
Teichoic acid and polysaccharide are also known to participate in the binding of
bacteriophages to some gram+ bacteria

b. Polysaccharides - composed of neutral and acidic sugars

ex of neutral sugars- raffinose, arabinose

2. GRAM NEGATIVE
- starts from the outer membrane
- convoluted, wrinkled, undulated membrane

composed of:

1. outer layer - phospholipids

2. proteins (porins) - matrix proteins; transport receptor factors(receptor for
bacteriophage)(small molecular weight for H2O soluble substances)
3. lipopolysaccharides - heat stable; lethally toxic pyrogenic

composed of:

1.
1. Lipid A - toxic and complex lipid; endotoxin of gram- bacteria
2 types of toxin
1. endotoxin - lipolysaccharide complex; needs lysis to release toxins; not
freely discharged
2. exotoxin - excreted from microorganisms; freely discharged

2. polysaccharides - major surface antigen of the gram- bacterial cell; a.k.a. O antigen

FXN of LIPOPROTEINS:

stabilizes of outer membrane and anchors it to the peptidoglycan layer

FXN OF OUTER LAYER:

prevents leakage of periplasmic proteins

protects the cell from bile salts and hydrolytic enzymes
 hinders the penetration of larger molecules hence, the relative resisitance of gram-
bacteria to number of antibiotics

STAINS
1. Gram's stain - most common
fixation - smear will stick to the slide

RGT PURPOSE TIME GM+ GM-

Crystal violet initial stain 1min violet violet

dirty
Gram's iodine mordant 1 min dirty bluish brown bluish
brown

remains violet (closes

Acetone
decolorizer 30 secs-1 min pores because colorless
alcohol
dehydrated)

counter stain or
Safranine 1 min still violet red
final stain

2. Acid fast staining technique or AFB (Bacillus) technique

ARTIFICALLY and NATURALLY OCCURRING MICROORGANISMS

1. artificially produced
protoplast - wall-less; application of lysozyme that dissolves cell wall; gram =
spheroplast - gram- wall-less
penicillin - destroys cell wall

2. naturally ocurring
mycoplasma - wall-less; protectd by osmolysis by adapting a parasitic existence; intracellular;
absorbs sterol and makes it their own sterol is not part of it

3. L-forms (Lister Institute)

either gram+/gram- that have almost lost all their cell wall during development or growth
in pus-filled wounds
most recurring infections caused by this

anti cell wall agent:

lysozyme - or muramidase; in phagocytes, tears, saliva, breast milk

 effect: catalyzes the hydrolysis of bonds between sugars in the polysaccharide chain
of the peptidoglycan

penicillin - inhibits peptide bridge formation

CELL MEMBRANE

thin structure adherent to the cell wall

comprises 30% cell weight
composition: 60% protein and 40% lipids

cell membrane difference between prokaryotes and eukaryotes: presence of esterols in

prokaryotes

FXNS:
A. selective permeability and transport of molecules in and out of the cell
cell membrane is impermeable to hydrophilic substances; hydrophobic layer around the
membrane

o acts as an osmotic link and osmotic barrier

o
permeases- protein enzymes; present in cell membrane and facilitate the
transport of molecules across the membranes; binds to the substance and
carries the substance inside the cell

General transport mechanisms:

1. active transport - uses energy in the form of ATP; specific solutes against a gradient low
to high concentration
2. passive transport - high to low concentration, no energy needed
1. simple diffusion - small molecules passively pass through cell membrane from H
to L (ex CO2, dissolved oxygen)
2. osmosis - diffusion but water ang mu pass
3. facilitated diffusion - no energy is needed; involves protein-membrane carriers (ex
permeases) found on the cell membrane - binds to substance and is actively
pumped inside the cell
3. group translocation - vectorial metabolism; facilitates the net uptake of sugars (ex
glucose, mannose); phosphorylated after passing the cell membrane (ex glucose -
glucose-6-phosphate); facilitates two processes: transport and metabolism
4. endocytosis
1. phagocytosis - solids
2. pinocytosis - liquids
in the case of oil drops, immediately transported inside the cell, do not depend of
being phagocytosed
 B. electron transport and oxidative phosphorylation
cytochromes and other enzymes in the respiratory chain are usually located in the
cytoplasmic membrane

C. Excretion of hydrolytic enzymes

bacillus. how does it eat?
macromolecules such as proteins polysacch cannot be immediately eaten by
make hydrolytic enzyme that will be excreted to the environment to hydrolyse the substances
present in the environment
once degraded, can be fused through cell membrane and thus eaten

MESOSOME
connective point to the DNA; involved in binary fission
membrane-associated cytoplasmic sacs that contain whorled, lamellar, tubluar or vesicular
structures that are associated with division septa; connected to DNA(nuclear body)
cell wall has invaginations(wot?) under this is a sac that contained whorled structures that are
connected to the DNA which is the nuclear body (comprised only of a single DNA that lies
freely in the cytoplasm)
where division of the nucleus starts; connected to division septa that divides the microorganism
later on into two; both take a part of the DNA
Binary Fission - how microorganism divide
[insert picture]

add ons:
APPENDAGES
1. Flagella - thread like; thinner than cilia
- made of flagellin (protein sub-unit) - for motility
- virulence factor
parts:

basal body or basal granule - anchored to the cell wall

basal hook - facilitates movement; enables flagella to move 360degrees
filament - lashing part; like a propeller; largest part of flagellum

how to know if motile:

1. microscopically:
1. wet mount
2. hanging drop - uses concavity slide; upon focusing under microscope, will be
known if it is moving kay wa man naipit
if not motile, unflagellated - only quivering, Brownian movement due to the
bombardment of molecules found in the suspending medium
2. macroscopically - kadtong ihumol sa semisolid medium nya kung mulapad then motile
 3. dark-filled microscopy - makes use of a dark-filled microscope

governed by chemotactic factors: (eg food)

positive chemotaxis - toward an attractant; counterclockwise

negative chemotaxis - away from a deterrent
aerotaxis - towards optimal oxygen concentration (especially aerobic microorganisms)
phototaxis - towards light (especially photosynthetic microorganisms)

sensoy transduction - mechanism by which a change in cell behavior is brought about in

response to a change in the environment; allows the cell to move; responsible for chemotaxis etc

Factors affecting flagellation:

composition of the medium (solid, semisolid, liquid)

pH of the medium (7.2 to 7.6 for medically important bacteria and 7 to 8 in general)
liquid or solid state of the medium

Types of Antigen

O-antigen - body; somatic

K-antigen - capsule
H-antigen - from flagella (Hauch)

o Pi factor (virulence factor) derived from the inner part of the composition of the
flagella

from serological test from serum then add reagent that will react with body, capsule or flagella
plasma - from anticoagulated blood
serum - coagulated blood, clear liquid

1.A. Axial filament - endoflagellum, periplasmic flagella; covered with outersheath

inside flagella
[insert picture]

2. Pilus/Fimbria - hairlike microfibrils; shorter, thinner than flagellum; important in

medicine since it transfers resistance
- virulence factor
- 0.004 to 0.008 in diameter
- 0.5 to 2 micrometer in length
- connects to another microorganism and then transfers its genetic material to
it; serves as a bridge

1. ordinary pili - adherence
2. sex pili - attachment for microorganism during conjugation

other types:

1. adhesin - or bacteriocinogens; adherence factor; colonization antigen
2. evasin - to evade phagocytosis since pili is a virulence factor
3. aggressin - leukocidal (kills WBC)
4. lectins - allow cells to bind to specific sugars on cell surfaces

1. Nuclear body or nucleoids - not called nucleus because it does not have any nuclear
membrane in prokaryotes
DNA fibril that runs parallel to the axis of the cell
2. Plasmids - can be differentiated from nucleoids since they are self replicating circles of DNA;
extrachromosal genetic elements so not connected to the nuclear body

conjugative plasmids - genes for carrying their transfer to another cell

dissimilation plasmids - genes that call for enzymes that catalyze the catabolism of
certain unusual sugars and hydrocarbons; some use unusual substances like hydrocarbons
of petroleum as source of carbon
bacteriocynogenic plasmids - contains the genes for synthesis of bacteriocins(toxic
proteins that kill bacteria)
R-factor or R-genes (resistance factor) - resistance to certain types of antibiotics
transposon - pieces of DNA; move readily from one area to another

3. Ribosomes - consists of 30% protein and 70% RNA; for protein synthesis scaattered
throughout the cytoplasm of the cell; look like sand; impart granular appearance to the cytoplasm
4. Endospores - highly refractile body formed within the vegetative cell at a certain stage of
growth; formed when there is absence of nitrogen or carbon in the medium; thermoresistant; !no
sporulated na rounded
in fungi for survival and reproduction
5. Inclusions
A. granules - food reserves

o metachromatic granules - stored from of polyphosphates; usually stain red with
blue dyes (methylene blue) ex: Corynebacterium diphtheriae; collectively called
volutin granules or polar granules
o
called Babes Ernst granules in Corynebacterium diptherae
o polysaccharide granules - consists of starch and glycogen; be demonstrated by
the use of plain iodine; glycogen will appear reddish brown and starch will appear
blue
 o lipid granules - unique to bacteria; polymer (poly-beta-hydroxybutyric acid);
demonstrated by fat soluble dyes (most common: Sudan dye); microbacterium has
lots of lipid granules, that is why very difficult to stain so gamiton ang Acid fast
stain
o sulfur granules - act as energy source of microorganism; possessed by sulfur
bacteria from genus Thiobacillus; deposit sulfur granules into the cytoplasm
o carboxysomes - polyhedral or hexagonal inclusions that contain the enzyme:
Ribulose-1,5-diphosphate carboxylase; ex nitribacteria, cyanobacteria

B. gas vacuoles - found in the cytoplasm of microorganism; appear as hollow cavities especially
on aquatic prokaryotes ex cyanobacteria ; fxn: maintain buoyancy

MICROBIAL METABOLISM
[28:39]
Microbial growth requires the polymerization of biochemical building blocks into proteins,
nucleic acids, polysacch, lipids
building blocks must come free form from the growth medium or synthesized from the organism
itself
when it comes from the organism itself, it emanates from a process called metabolism
metabolism - sum of all chemical reactions that usually occur in a living organism, energy
balancing act
metabolite - any molecule that is a nutrient, end product or inermediate
2 rxns:
anabolism - synthesis of macromolecules or complex substances
endergonic bc energy is needed and used
catabolism - degredative, exergonic rxn, energy yielding;
breakdown of complex substances to simpler substances

common source of energy of bacteria: GLUCOSE, simplest form of polysaccharide

energy source among bacteria could be:

o glucose
o sulfur
o gas in the form of Hydrogen
o light

Through the process of oxidation, energy (ATP) is produced by the release hydrogen ions or
electrons. Not all energy produced are absorbed by the microorganism, some are released into
the environment in the form of heat while some are carried by electron carriers such as NAD,
NADP or FAD
glucose is the main carbohydrate source
depending on the enzymatic composition and presence or absence of oxygen,

Degradation of glucose occurs in three pathways:

1. Embden-Meyerhoff-Parnas Pathway
- mixed acid fermenation
- fermentation bc in the absence of oxygen
- anaerobic glycolytic pathway
- usually used by anaerobic microorganisms

a. glucose is phosphorylated to form glucose-6-phosphate by the enzyme hexokinase

b. turned to fructose-6-phosphate by the enzyme isomerase
c. turned to fructose-1,6-diphosphate by the enzyme phosphofructokinase
d. converted to Glyceraldehyde-3-Phosphate by the enzyme aldolase
e. then converted to pyruvic acid which is the intermediate hydrogen acceptor which in turn is
oxidized to form acids
ex: lactic acids from sodium lactate
Acetyl CoA to Acetic Acid

!if bacteria has the ability and enzymes to degrade the acids it will do so, such as:
 formic acid to H and CO2
Acetic acid to butyric acid to buryl alcohol
acetaldehyde to ethyl alcohol

Starts with: phosphorylation of glucose

Intermediate H acceptor: Pyruvic acid
Final hydrogen acceptor: organic compounds or salts
Final product: acids (and simple compounds and substances, if bacteria is capable)

2. Entner Duodoroff Pathway

a. also starts with the phosphorylation of glucose
b. converted to Glucono-Lactone-6-PO4
 G-6-P is not converted to F-1,6-DP since microorganism does not have necessary enzymes to
do so
c. converted to 6-Phosphogluconate
d. converted to 2-Keto-3-Deoxy-6-Phosphogluconate
e. converted Pyruvic Acid
aerobic microorganisms lack necessary hydrogenase enzymes to finally break down, no acid is
produced
pyruvic acid has to transfer hydrogen ions into the Krebs cycle which combines with detrimental
hydrogen to form water

Starts with: phosphorylation of glucose

Intermediate H acceptor: Pyruvic acid
Final hydrogen acceptor: oxygen in the Kreb's cycle
Final product: water

3. Warburg Dickens Hexose Monophosphate Pathway

- hybrid between the aerobic and anaerobic pathways
- used by aerotolerants or facultative microorganisms
-basically anaerobic but can tolerate small amounts of oxygen
- sometimes called the Pentose Pathway because of the presence of Ribulose-5-Phosphate in its
process
Starts with: phosphorylation of glucose
Intermediate H acceptor: Pyruvic acid
Final hydrogen acceptor: oxygen in the Kreb's cycle and organic salts and compounds
Final products: water and mixed compounds

GROWTH REQ

Physical requirements: (OTHRO)

1. Oxygen
2 types of microorganisms based on oxygen requirements

aerobic - need oxygen

o obligate - only survive in the presence of oxygen

- requires 20-21% oxygen in 1atm and 0.3% CO2

o facultative - basically anaerobic but can survive in the presence of small amounts
of oxygen
anaerobic - unable to use oxygen as an electron acceptor

- 5-10% hydrogen, 5-10% CO2 and 80-90% nitrogen

others:

microaerophilic, microphilic - requires less than 1atm of oxygen, 0.2atm ra ang

kahinanglan = 5-10% oxygen

- increased CO2 usually 8-10%

capnophilic or capneic - organisms grow best with higher CO2 concentration only 15%
oxygen

2. Temperature

Mesophilic - 37C or (25-45C)

Psichorophilic - survive that 0 to 10C; below 10C (0-24C)
Thermophilic - survive above 45C

3. Hydrogen ion concentration or pH

most bacteria grow at a pH of 6-8
Bacteria of medical importance: 7.2 - 7.6
fungi are acidic
grow in alkaline pH ~9.6 Vivio cholerae

acidophils pH of 0.1-5.4
neutrophil - pH of 5.5-7
alkaliphils - pH of 8-11.5

4. Radiation
in the form of light
5. Osmotic pressure

plasmolysis - water from inside the cell will go outside the cell; shrinking of cell;
hypertonic
plasmoptysis - in hypotonic, solute conc is higher inside the cell than the outside; cell
will burst

also:

halophilic - organism that require high salt concentration only one of medical
importance: Vivio cholerae

BACTERIAL GROWTH

[1:13:09]
major function of any microbial cell is mainly growth
growth - orderly increase in mass or number of all components of the cell
- via simple binary fission
- logarithmic/exponential ila growth, people kay arithmethic

Generation time - time required to accomplish binary fission;

*how to estimate generation time?
by >Actual Bacterial Count;
>Measuring the rate of enzymatic activity
Growth rate constant - factor that expresses the average number of times the cell will divide in
a given time period

generation time vs growth rate constant:

how much time will the microorganism become 2? vs how many times will the cell divide in
1min? or 5mins?

Microbial growth can be measured by:

1. cell concentration - number of cells per unit volume of culture; viable cell count
2. cell density - refers to the dry weight of the cell or cells per unit volume of culture
- now replaced by photoelectric measurement, nitrogen determination or
centrifugation in special vessels

Can also be measured by:

Bacterial Growth Curve
you get a sample (loophole), transfer into a new or fresh culture medium and place it there
will not immediately grow, thus Lag Phase
A. Lag Phase, Physiological Youth, Phase of Rejuvenescence

dile zero magsugod ang Lag Phase kay naa may gamay na microorganisms
growth rate: 0
represents a period during which the cells, depleted of metabolites and enzymes as a
result of unfavorable conditions that was obtained at the end of their previous culture
history, adapt to their new environment
increase in:

o metabolic activity
o bacterial RNA and protein synthesis
o bacterial cell size
o macromolecular components
o susceptibility to physical and chemical agents

when adapted to new environment, they will now increase in activity so Lag phase will start to
go up, slowly not abrupt

B. Exponential Phase

constant growth rate

represents the peak of growth activity in the culture medium, cells are dividing at a
constant rate multiplying by geometric progression
afterwards:

 o either one or more nutrients in the medium will become exhausted
o toxic metabolites accumulate and impede growth, bacteria at this point especially
is susceptible to antibiotics

C. Maximum Stationary Growth Phase

0 growth
some will start to die kay exhausted na

balanced by the formation of new cells through growth and division

plateau
the number of new cells is balanced by dead cells
increased growth rate as a result of the detererious effect on the culture of the following:

o accumulation of waste products
o exhaustion of nutrients, especially carbon, nitrogen sources
o change in pH - 7.2-7.4 will become acidic because of metabolic by products

D. Death Phase or Phase of Decline

growth rate decliing

occurs when majority of the cells have died and the death rate has decreased drastically
small number of survivors may persist for months or even years
a few cells growing at the expense of released nutrients from cells that died and lysed