COURSE OUTLINE: MIDTERMS procedure; so appear purple

● Retain crystal violet dye in Gram staining

1. CELL WALL
procedure; so appear purple
2. MICROBIAL NUTRITION AND GROWTH
● no outer membrane
3. NORMAL FLORA
REFERENCE BOOK

CELL WALL
PROKARYOTIC CELL WALLS
● Provide structure and shape and protect cell from
osmotic forces
● Assist some cells in attaching to other cells or in
eluding antimicrobial drugs
● Not present in animal cells, so can target cell wall of
bacteria with antibiotics
● Bacteria and archaea have different cell wall
Figure C. Gram positive cell wall
chemistry

BACTERIAL CELL WALLS

● Most have cell wall composed of peptidoglycan
● Peptidoglycan is composed of sugars, NAG
(N-acetylglucosamine), and NAM
(N-acetylmuramic)
○ this networks consists of repeating
disaccharide connected by polypeptides to
form a lattice that surrounds and protects
the entire cell
● Chains of NAG and NAM attached to other chains
by tetrapeptide crossbridges
○ Bridges may be covalently bonded to one
another
○ Bridges may be held together by short
connecting chains of amino acids
● Scientists describe two basic types of bacterial cell
walls: Gram-positive and Gram-negative Figure D. Structure of a gram positive cell wall

wall teichoic acid lipoteichoic acid

definition wall teichoic acids are are teichoic acids

the teichoic acids attached to lipid in
covalently attached to the cytoplasmic
peptidoglycan membrane

attachment covalently attached to covalently linked to

peptidoglycan the lipid in the
cytoplasmic
Figure A. External Structures of Prokaryotic Cells membrane

function cell shape determination, contributes a

regulation of cell division, negative charge to
pathogenesis, antibiotic the cell wall and acts
resistance and other as a receptor
fundamental aspects of molecule for some
gram-positive bacterial gram-positive
physiology bacteriophage

GRAM-NEGATIVE CELL WALLS

● Have only a thin layer of peptidoglycan
● Bilayer membrane outside the peptidoglycan
contains phospholipids, proteins, and
Figure B. External Structures of Prokaryotic Cells
lipopolysaccharide (LPS)
GRAM-POSITIVE CELL WALLS ● May be impediment to the treatment of disease
● Relatively thick layer of peptidoglycan ● Appear pink following Gram staining procedure
● Contain unique polyalcohols called teichoic acids
● Some covalently linked to lipids, forming lipoteichoic
acids that anchor peptidoglycan to cell membrane
● Retain crystal violet dye in Gram staining

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 Figure F. Process of Gram staining procedure

Figure E. Gram Negative Cell wall mordant- chemical that fixes in place a dye already
present

FUNCTIONS OF LIPOPOLYSACCHARIDES >All bacilli are gram-negative except Corynebacterium,

● The core polysaccharide usually contains charged Mycobacterium, aerobic spore-formers (Bacillus), and
sugars and phosphate, LPS contributes to the anaerobic spore-formers (Clostridium).
negative charge on the bacterial surface >All cocci are gram-positive except Neisseria, Veillonella,
● Bacterial attachment to surfaces and biofilm and Branhamella.
formation
● it aids in creating a permeability barrier to restrict
the entry of bile salts, antibiotics, and other toxic
substances that might kill or injure the bacterium
● The O side chain of LPS are also called the O
antigen because it elicits an immune response
● G negative bacteria are able to rapidly change the
antigenic nature of their O side chains, thus
escaping host defenses

Figure G. Expected results in each reagent

MORPHOLOGIES

Figure E.

LIPID A
● Hydrophobic group of lipopolysaccharide, covers
the surface of most Gram-negative bacteria.
● Lipopolysaccharide, known as endotoxin, can cause
fatal disease like sepsis syndrome.
● Lipopolysaccharide, known as endotoxin, can cause
fatal disease like sepsis syndrome.
ACID-FAST BACTERIA
GRAM STAINING PROCEDURE
ACID FAST CELL WALL OF MYCOBACTERIUM
● Thick peptidoglycan linked to arabinogalactan
(D-arabinose and D-galactose) which is then linked
to high-molecular-weight mycolic acids consisting of

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 free lipids, phenolic glycolipids, and peptide
glycolipids MICROBIAL NUTRITION AND
● Other glycolipids include lipoarabinomannan and
phosphatidylinositol mannosides (PIM).
GROWTH
● Microbial growth – an increase in a population of
● Like the outer membrane of the gram-negative cell
microbes rather than an increase in size of an
wall, porins are required to transport small
individual
hydrophilic molecules through the outer membrane
● Result of microbial growth is discrete colony –
of the acid-fast cell wall.
an aggregation of cells arising from single parent
● Because of its unique cell wall, when it is stained by
cell
the acid-fast procedure, it will resist decolorization
● Reproduction results in growth
with acid-alcohol and stain red, the color of the
● Organisms use a variety of nutrients for their energy
initial stain, carbol fuchsin.
needs and to build organic molecules and cellular
○ With the exception of a very few other
structures
acid-fast bacteria such as Nocardia, all
● Most common nutrients – those containing
other bacteria will be decolorized and stain
necessary elements such as carbon, oxygen,
blue, the color of the methylene blue
nitrogen, and hydrogen
counterstain
● Microbes obtain nutrients from variety of sources

element propor function

tion

carbon 50% basic structural component of

organic compounds

Oxygen 20% component of many organic and

inorganic compounds ; O2 is the final
electron acceptor in aerobic
respiration

Nitrogen 14% constituent of amino acids and

nucleotides
(1) Mycolic Acid - waxy substance in cell wall
(2) Arabinogalactan
(3) Peptidoglycan Hydrogen 8% constituent of organic compounds;
(4) cytoplasmic membrane electrons of hydrogen atoms are
used in redox reactions

phosphorus 3% As phosphate (PO4 2-), a constituent

of nucleic acids, phospholipids, some
coenzymes and ATP

ACID-FAST STAINING Sodium 1% cation used to maintain electrical

balance of cells

Sulfur 1% constituent of amino acids cysteine

and methionine; disulfide bonds are
important for tertiary structure of
proteins

Potassium 1% Inorganic carbon involved in

maintaining electrical balance inside
cells; cofactor for some enzymes,
1. Prepare and fix the specimen smear prior to including some involved in protein
staining. synthesis
2. Place a small strip of blotting or filter paper over the
top of the specimen, and place the slide over a Chlorine 0.5% principal inorganic anion involved in
boiling hot water bath on a mesh surface. maintaining electrical balance of cells
3. Cover the filter paper with the primary stain,
carbolfuchsin. Leave the slide on the water bath for Magnesium 0.5% Component of chlorophyll; cofactor
3 to 5 minutes. Continue to apply stain if the filter for some enzymes
paper begins to dry.
4. Remove the filter paper and rinse the slide with
water until the solution runs clear.
SOURCES OF CARBON, ENERGY AND
5. Run acid-alcohol decolorizer over the slide for
approximately 10 to 15 seconds. ELECTRONS
6. Rinse the slide with water.
7. Cover the smear with the secondary or BASED ON SOURCE OF CARBON
counterstain, methylene blue, for 1 minute.
8. Gently rinse the slide with water. AUTOTROPHS
9. Blot the slide dry with paper. ● use an inorganic source of carbon (carbon dioxide)

HETEROTROPHS

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 ● catabolize reduced organic molecules (proteins,
carbohydrates, amino acids, and fatty acids)
BASED ON USE OF CHEMICALS OR LIGHT AS A
SOURCE OF ENERGY
CHEMOTROPHS
● acquire energy from redox reactions involving
inorganic and organic chemicals
PHOTOTROPHS
● Phototrophs use light as their energy source
COMBINATION
OXYGEN REQUIREMENTS
FOUR TOXIC FORMS OF OXYGEN
SINGLET OXYGEN
● molecular oxygen with electrons boosted to higher
energy state
● Occurs during photosynthesis so phototrophic
organisms have carotenoids that remove the
excess energy of singlet oxygen
SUPEROXIDE RADICALS
● some form during incomplete reduction of oxygen in
aerobic and anaerobic respiration
○ So reactive that aerobes produce
superoxide dismutases to detoxify them
○ Anaerobes lack superoxide dismutase and
die as a result of oxidizing reactions of
superoxide radicals formed in presence of
oxygen
PEROXIDE ANION
● formed during reactions catalyzed by superoxide
dismutase and other reactions
○ Aerobes contain either catalase or
peroxidase to detoxify peroxide anion
○ Obligate anaerobes either lack both
enzymes or have only a small amount of
each
HYDROXYL RADICAL
● results from ionizing radiation and from incomplete
reduction of hydrogen peroxide
● The most reactive of the four toxic forms of oxygen
● Not a threat to aerobes due to action of catalase
and peroxidase
TERMINOLOGIES
AEROBES
● undergo aerobic respiration
ANAEROBES
● do not use aerobic metabolism
FACULTATIVE ANAEROBES
● can maintain life via fermentation or anaerobic
respiration or by aerobic respiration
AEROTOLERANT ANAEROBES
● do not use aerobic metabolism but have some
enzymes that detoxify oxygen’s poisonous forms
MICROAEROPHILES
● aerobes that require oxygen levels from 2 to 10%
and have a limited ability to detoxify hydrogen
peroxide and superoxide radicals
NITROGEN REQUIREMENTS
● Anabolism often ceases due to insufficient nitrogen
needed for proteins and nucleotides
● Nitrogen acquired from organic and inorganic
nutrients, plus all cells recycle nitrogen from amino
acids and nucleotides
● The reduction of nitrogen gas to ammonia (nitrogen
fixation) by certain bacteria is essential to life on
Earth because nitrogen is made available in a
usable form
OTHER CHEMICAL REQUIREMENTS
● Phosphorus is a component of phospholipid
membranes, DNA, RNA, ATP, and some proteins
● Sulfur is a component of sulfur-containing amino
acids, disulfide bonds critical to tertiary structure of
proteins, and in vitamins (thiamin and biotin)
● Trace elements – only required in small amounts;
usually found in sufficient quantities in tap water
● Growth factors – necessary organic chemicals that
cannot be synthesized by certain organisms
(vitamins, certain amino acids, purines, pyrimidines,
cholesterol, NADH, and heme)
PHYSICAL REQUIREMENTS
TEMPERATURE
● Effect of temperature on proteins
● Effect of temperature on lipid-containing
membranes of cells and organelles
○ If too low, membranes become rigid and
fragile
○ If too high, membranes become too fluid
and cannot contain the cell or organelle
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 ■ -Their membranes and enzymes
depend on this pressure to
maintain their three-dimensional,
functional shape

ASSOCIATIONS
● Organisms live in association with different species
○ Antagonistic relationships
○ Synergistic relationships
○ Symbiotic relationships

BIOFILMS
● Complex relationships among numerous
microorganisms
● Develop an extracellular matrix that adheres cells to
one another, allows attachment to a substrate,
pH sequesters nutrients, and may protect individuals in
● Organisms sensitive to changes in acidity because the biofilm
H+ and OH- interfere with H bonding in proteins and ● Form on surfaces often as a result of quorum
nucleic acids sensing
● Neutrophils are bacteria and protozoa that grow ● Many microorganisms become more harmful when
best in a narrow range around neutral pH (6.5-7.5) part of a biofilm
● Acidophiles are bacteria and fungi that grow best in
acidic habitats
○ Acidic waste products can help preserve CULTURING MICROORGANISMS
foods by preventing further microbial ● Inoculum introduced into medium (broth or solid)
growth ○ Environmental specimens
● Alkaliphiles live in alkaline soils and water up to pH ○ Clinical specimens
11.5 ○ Stored specimens
● Culture – refers to act of cultivating microorganisms
PHYSICAL EFFECTS OF WATER or the microorganisms that are cultivated
● Microbes require water to dissolve enzymes and
nutrients required in metabolism
● Water is important reactant in many metabolic
reactions
● Most cells die in absence of water
○ Some have cell walls that retain water
○ Endospores and cysts cease most
metabolic activity in a dry environment for
years
● Two physical effects of water
○ Osmotic pressure
■ The pressure exerted on a
semipermeable membrane by a
solution containing solutes that
cannot freely cross membrane;
related to concentration of
dissolved molecules and ions in a
solution
■ Hypotonic solutions have lower
solute concentrations; cells
placed in these solutions will
swell and burst
■ Hypertonic solutions have greater
solute concentrations; cells
placed in these solutions will
undergo crenation
● This effect helps
preserve some foods
■ Restricts organisms to certain
environments
● Obligate halophiles –
grow in up to 30% salt
● Facultative halophiles –
can tolerate high salt
concentrations
○ Hydrostatic pressure
■ Water exerts pressure in
proportion to its depth -For every
additional 10 m of depth, water
pressure increases 1 atm
■ Organisms that live under
extreme pressure are barophiles

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CLINICAL SPECIMENS AND THE METHODS USED NORMAL FLORA IN DIFFERENT SITES OF BODY
TO COLLECT THEM

NORMAL FLORA
MICROBIAL ECOLOGY
● the study of the relationships between
microorganisms and their environment

NORMAL FLORA
● consists of the group of organisms that inhibit the
body of a normal health individual in the community

SKIN
● in constant contact with the environment (most
exposed to microorganisms)
● factors to eliminate non-resident flora from the skin
○ lysozyme in the skin
TYPES OF NORMAL FLORA ○ acidic ph (sweat)
○ free fatty acids in sebaceous secretions
RESIDENT FLORA ● constant sloughing off of the skin
● organisms that are relatively of fixed types and ● 3 regions of the skin
regularly found in a given area of the body at a ○ axillary,perineum and toe webs - high
given age moisture levels, high body temperature
and high levels of surface lipids and it has
TRANSIENT FLORA more microorganisms that are gram
negative
● are those that inhabit the skin and mucous
○ hand, face trunk
membrane beneficial for hours, days, or weeks and
○ upper arms and legs
are derived from the environment
STAPHYLOCOCCUS EPIDERMIDIS
ADVANTAGES OF NORMAL FLORA
● normal flora protects the body’s organs and ● major skin inhabitant
systems that are in direct contact with the external ● compromising approximately 90% of resident
environment and are therefore subject to the attach aerobic rods
of invasive organisms
● they compete with the invasive organisms for STAPHYLOCOCCUS AUREUS
nutrients essential for their growth or by producing ● most commonly found in nose and perineum
substances that can kill them ○ in the nose, the number varies with age
● it synthesize important vitamins like B12 and K (greater in newborns than in adults
○ ex: normal intestinal flora secrete vitamin K
that is needed for the activity of some MICROCOCCI (MICROCOCCUS LUTEUS)
clotting factors ● accounts for 20%-80% of micrococci in the skin
● prevents pathogenic organisms by attaching and
penetrating the skin and other tissues by producing DIPHTHEROIDS (CORYNEFORMS)
Mucin ● lipophilic (axilla)
● normal flora in the intestines aid in the digestion of ● non lipophilic (hairless skin)
food by producing enzymes such as cellulase, ● anaerobic diphtheroids (propionibacterium acnes)
galactosidase, and glucosidase
● intestinal flora also help in the metabolism of
GRAM-NEGATIVE BACILLI
steroids
● example: enterobacter klebsiella, escherichia coli,
proteus
DISADVANTAGES OF NORMAL FLORA
● seen in most intertriginous areas such as toe webs
● production of disease if the individual becomes
and axilla
immunocompromised or if they change their usual
anatomic locations
● production of disease since most of them are NAIL FLORA
pathogens or opportunistic pathogens ● similar to that of the skin
● fungi may also be present (aspergillus, Penicillium,

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 Cladosporium, Mucor)

DIGESTIVE SYSTEM

OTHER SITES

body site flora names

upper Staphylococcus aureus

respiratory Haemophilus influenzae
tract Staphylococcus pneumoniae

Skin surface Staphylococcus aureus

Staphylococcus epidermidis
Propionibacterium acnes
Candida spp. (yeast)

Vagina Lactobacillus spp.

Candida albicans (yeast)
Trichomonas vaginalis (protozoan)

Large intestine Escherichia coli

Clostridium spp.
Proteus spp.
Klebsiella spp.
Lactobacillus spp.
Clostridium perfringens
Candida albicans (yeast)

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