Prokaryotes Eukaryotes

Small (5 micrometer) Larger (up to 40 micrometer)

No nucleus present
Few organelles present, no membrane-bound
Rigid cell wall formed from glycoprotein (mainly
murein); all have cell walls
70S Ribosomes
Chlorophyll scattered in cytoplasm
Cell division: binary fission
Transfer of DNA fragments only (Conjugation)
No true chromosomes instead only Plastids
Usually unicellular (some cyanobacteria
multicellular)
Has true nucleus
Many organelles present; some have double
membrane; others single membrane
Fungi: Rigid cell wall from polysaccharide
(Chitin)
Plant: Rigid cell wall (cellulose)
Animals: No cell wall (only some has CW)
80S Ribosomes
Chlorophyll found in chloroplast in plants
Cell division: Mitosis
Has Golgi apparatus, mitochondrion, lysosomes,
peroxisomes
More than one chromosome
Usually, multicellular

• Microscopic and unicellular

• Smaller than 100 micrometers
• Propagation with solid media colonies (10x8 cells)
• Parts:
a) Plasma membrane/Cytoplasmic membrane
- Periphery, elastic, semi-permeable, phospholipid bilayer and proteins
b) Cell Wall
- External to plasma membrane, rigid, porous, maintains cell shape
c) Cell Envelope
- Collective term for cell wall & plasma membrane
d) Cytoplasm
- Aqueous part packed with ribosomes and proteins
e) Cytoskeletal network
- Maintains cell shape & for cell division and spore formation
f) Inclusion bodies
- Spores, volutin granules, lipids, and glycogen
g) Nucleoid/ Nuclear Material
- Double stranded DNA (genetic info)
- Plasmids: extrachromosomal DNA
- Haploid
- Associated with NAP (Nuclear Associated Proteins)
h) Capsule
- Gelatinous layer outside cell wall
i) Flagella
- Outward projections; protein-based filamentous appendages
- Organs of locomotion
j) Fimbriae
- Hair-like structures
k) Pili
- Mediate adhesion between cells tip through a specific receptor-ligand
l) Ribosomes
- Smaller than ribosomes of eukaryotic cells
- 70S – with subunits 30S and 50S
m) Polyribosomes
- Single mRNA with several ribosomes
- Peptidoglycan/ mucopeptide/ murein: main component that give strength
- N-acetylglucosamine & N- acetylmuramic acid
- Teichoic acid & lipoteichoic acid
- Gram (+) bacteria – thicker wall

- Outside of peptidoglycan
- Gram (-) bacteria
- Made of proteins with porins; semi-permeable & attachment
- Protects cell wall from effects of lysozymes & ingress of antibiotics
- Component – Lipid A – core structure - endotoxin
- Breakdown; shock syndrome & other systemic conditions
- Mycobacteria (Tuberculosis and Leprosy)
→ Peptidoglycan (arabinogalactan); Mycolic acid (beta-hydroxy fatty acid); forms
a hydrophobic external layer barrier
→ Acid-staining (Zhiel-Neelsen & Phenol-auramine Method)

- Capsule functions: protect against phagocytosis, lytic action of complement,

bacteriophage invasion and desiccation
- microcapsule
- Slime: amorphous, viscid, colloid secreted by cells
▪ Culture media: watery, sticky mucoid characteristics
- S-layer: structural protein same function with capsule
- biofilm

- Flagella:
o filamentous appendages; single or more per cell
o for locomotion – Escherichia coli – tumbling
o arrangement-
▪ peritrichous – sides or lateral
▪ polar – monotrichous; amphitrichous; lophotrichousto or another bacteria
w/o

- Fimbriae: more numerous and shorter

o Adhesion between bacteria and host cells
- Pili: for genetic exchange between bacteria
o Sex Pili: longer and attaches to other bacteria that lacks appendage
▪ Initiates conjugation
▪ Acts as receptor for bacteriophage

- Vegetive or growing
- Stationary or dead
- Spore formation: dormancy
 - Bacillus and clostridium
- Endospore: highly resistant resting phase
- Resists with drying, heat and disinfectants
- Killed with temperature at 100-120 degrees Celsius or more for 10-20 minutes
- Location: terminal, subterminal, central
- “Germination:” reactivation of spores in response with some stimuli
- Exospores or conidia

1. Pleomorphism – variation of size, shape, and form during growth; Streptobacillus

moniliformisn yersinia pestis

2. Ivolution – abnormal cells or degenerative cells; non-viable

3. Protoplast (free) – if all cell wall removed
4. Sheroplast – weak cell wall
5. L-forms bacteria - cell wall deficient
Growth, Survival, Death of Microorganisms
• Bacterial growth and multiplication is of practical value in the detection and identification
of pathogens.
• Bacterial growth involves both in increase In the size and number of microorganisms.
• Growth results from bacterial reproduction due to binary fission. In the laboratory, growth
can be seen in 3 forms:
1.) development of colonies
2.) transformation of a clear broth medium to a turbid suspension
3.) biofilm production
Bacterial Growth Curve-Phases:
1. Lag phase
- Number of cells present appears to remain constant
- Metabolite: depleted cells adapt to new environment
- “flat period”
2. Exponential or log phase
- Increase in the cell number becomes detectable and rate accelerates rapidly until it is
established at the maximum achievable rate
- Cells at this stage are generally susceptible to antibiotics
3. Stationary phase
- Cells exhaust essential nutrients or accumulate toxic products; growth slows down
4. Death phase
- Cells die due to toxic products
Practical Importance of Growth Curve:
- Actively growing cells are more vulnerable to conditions that disrupt cell
metabolism and binary fission
- Microbes in Exponential phase are more vulnerable to heat and disinfectants
- Microbial cells produced during exponential phase are more numerous and virulent
Bacterial Cultivation
- Propagation of bacteria by providing the proper environmental conditions
Requirements for Growth:
1. Organic matter- carbon, hydrogen, nitrogen, oxygen, phosphorus, sulfur
2. Inorganic ions- K, Na, Fe, Mg, Ca, Cl (required for enzymatic catalysis and to maintain
chemical gradient across the cell membrane
3. Energy- Derived from ATP and proton motive force
- Phototrophic vs Chemotrophic org.
- Organotrophic vs Lithotrophic org.
Factors that Control Microbial Growth:
1. Nutrients
2. Temperature
3. pH
4. Moisture
5. Radiation
6. Gases
7. Other microorganisms
Nutrients
Source of Essential Nutrients:
a) Carbon sources- Proteins, carbohydrates, lipids, nucleic acids
- Heterotrophs
▪ “Hetero”- others “Troph”- to feed
Then
▪ Require performed organic compounds for growth
▪ Dependent on other life forms
- Autotrophs
▪ Have the special capacity to convert CO2 into organic compounds, this, they
are not nutritionally dependent on other living things
b) Nitrogen sources
 Nitrogen
79.1 ,

Oxygen
/
'

20 ,

Main reservoir for nitrogen is nitrogen gas (makes up 79% of the earth’s
-
atmosphere)
- An indispensable element to the structure of protein, DNA, RNA, ATP
- 5% of dry wt. of bacterial cell
- Ammonia- End product of all pathways for nitrogen assimilation
c) Sulfur source
- Sulfur is an essential component of some vitamins (vit.B1), amino acids(methionine,
cysteine), forms part of the structure of several coenzymes
- Rocks and sediments contain sulfate, sulfides and elemental sulfur
d) Phosphorus sources
- Main inorganic source of phosphorus is phosphate
- Phosphate is the key component of nucleic acids, ATP, coenzymes
e) Oxygen sources
- Oxygen is the major component of organic compounds (proteins, carbohydrates,
lipids)
- Free gaseous O2 makes up 20% of atmosphere

f) Hydrogen sources
- Hydrogen is a major element in all organic compounds
- Roles- Maintain pH, forms H2 bonds between Molecules serves as source of free
energy in oxidation
ILES 20 CW c
* ME SOPH
-

hot
Temperature * THERMOPHILES
cold
Ba 60C

A doc
* PSYCHROPHILES
Mesophiles MODRIC
Short exposure
of activity volcanic

* THER
- grow best between 20-40 C normally mesophiles
kid

- ex. Most bacterial pathogens

- Inhabit animals, plants, soil and water in temperate, subtropical and tropical regions
Thermophiles
- Grow best at high temperatures (>60 C)
- Ex. Bacillus stearothermophilus
- Live in soil and water assisted with volcanic activity and in those directly exposed to sun
- “heat loving”
Psychrophiles
- Grow best ar low temperature (<10 C)
- Ex. Flavobacterium species
- Inhabit snowfields, polar ice, deep ocean
- “Cold loving”
Thermodric
- Can survive short exposure to high temperature but are normally mesophiles
- Common contaminants of heated or pasteurized foods
- Ex. Giardia, Bacillus, Clostridium
Range of Temperature for Microbial Growth:
3 Cardial temperatures:
1. Minimum temperature- Lowest temperature that permits a microbe’s continued growth and
metabolism
2. Maximum temperature- Highest temperature at which growth and metabolism can proceed
3. Optimum temperature- Between minimum and maximum & promotes the fastest rate of
growth and metabolism

pH
- Degree of acidity or alkalinity solution: Hydrogen ion concentration
- Range 0-14
- Most organisms live or grow in habitats between pH 6 and 8
- Neutralophiles- grow best at pH 6-8
- Acidophiles- grow best as low as pH 3
6 8
NNEEUU#E0PtHtk$ ph
-

tf

3
ABB AAAA ⑧ AHH * Ph
98100
AM AB KAKA PHA LES pts
 - Alkaliphiles- grow best as high as pH 10
Gases
O2 and CO2 that most influence microbial growth
- Strict (Obligate) Aerobes
AEROBES
▪ Require O2 for growth
▪ Contain superoxide dismutase which protects them from toxic O2 (the
enzyme carries out the reaction that produces hydrogen peroxide which toxic
to cells but is destroyed by catalase or is oxidized by peroxide
▪ Ex. Pseudomonas aeruginosa
- Strict (Obligate) Anaerobes
▪ Require the complete absence of O2 ANAEROBES
▪ Killed by O2
▪ Lack superoxide dismutase, catalase and cytochrome-C oxidase (Enzymes
that destroy toxic products of O2)
▪ Instead of O2, they require another substance like hydrogen
▪ Outnumber aerobes 1000:1 in gut, 100:1 in the mouth
▪ Comprise 99% of total fecal flora
▪ Usually cause polymicrobial infections
▪ Foul-smelling
▪ Ex. Bacteroides fragilis
- Facultative Anaerobes
▪ Grow in the presence or absence of O2
▪ They shift from fermentative to respiratory metabolism in the presence of air
▪ Most pathogenic bacteria are facultative anaerobes
▪ Ex. E.coli, staph. Species
- Aerotolerant Anaerobes
▪ Have a fermentative metabolism with or without an O2 environment
▪ Ex. Clostridium perfringens
- Micro-aerophilic organisms
▪ Requires a reduced O2 level (5%)
▪ Ex. Campylobacter, Helicobacter
- Capnophilic organisms – Require high CO2 levels (ex. Neisseria Species)
- Dental carries are partly due to the complex actions of aerobic and anaerobic bacteria, and
most gingival infections consists of similar mixtures of oral bacteria that have invaded
damaged gum tissues
Microbial Associations
a) Symbiosis- 2 organisms living together in close partnership
Types:
1. Mutualism- Organism living in obligatory but mutually beneficial relationship
2. Commensalism- Commensal receives benefits while its cohabitant is neither harmed
nor benefited & Satellism- Classic commensalism (One member provides nutritional
or protective factors on the other)
3. Parasitism- Host organism provides the parasitic microbe with nutrients and habitat
& host is harmed
b) Non-Symbiosis- Organism are free-living, relationship not required for survival
Types:
1. Synergism- Participants cooperate to produce a result that none of them could do
alone
2. Antagonism- Members compete & some members are inhabited or destroyed by
others
Methods of Culturing Microorganisms
5 basic Techniques (5I’s)
1) Inoculation
2) Incubation
3) Isolation
4) Inspection
 .gs#Ei---E7
5) Identification
Inoculation
- Org. are introduced into a container or nutrient medium which provides an environment in
which they multiply
- The observable growth that appears in or on the medium is known aslast
culture
Isolation
- Separation of one species from another
- Discrete mound of cells is called a1-7
colony
Methods:
-- Streak plate method
- Loop dilution/ pour plate technique
=- Spread plate technique
Incubation
- Inoculated medium is placed in a temperature-controlled chamber to encourage
multiplication
Inspection
- Cultures are observed macroscopically for obvious growth characteristics; slides are made
to assess microscopic details
Identification
- To determine the type of microbe
Preparing specimens for microscopes:
1) Fresh, living preparations
2) Fixed, stained smears
Staining methods:
1) Negative
➢ dye does not stick to the specimen but settles around its outer boundary
➢ Cells are not stained because dyes(nigrosine, India ink) are negatively charged and
are repelled by negatively charged surface of cells
2) Positive
➢ Dye sticks to the specimen
a) Simple- single dye
b) Differential- use 2 different-colored dyes to distinguish cell types or parts
Differential staining:
a) Gram staining- sequential application of crystal violet (primary dye), gram’s iodine
(mordant), and alcohol rinse (decolorizer) and contrasting counterstain
- Gram (+)- Purple
- Gram (-)- Pink or red
b) Acid- fast stain
- Acid-fast bacteria- Pink
-

- Non-acid-fast bacteria- Blue

-

c) Special stains- endospore, capsule, flagella

Growth medium
1. Minimal essential growth medium
-
Nsa.
- Contains only the primary precursor compounds essential for growth
it

RN - Generation time- slow ng

2. Complex growth medium

-
Mad
- Contains most of the organic compound building blocks necessary for growth
fast - Generation time- faster
←
a-

- Fastidious bacteria are grown in this medium

3) Differential growth medium
-

- Contains a combination of nutrients and pH indicators to allow the visual distinction

Mft OWNS t

,
of bacteria Ex Bloop A
.
AR
HEMOLYSIS
TYPES
DISTINGUISH OF
 - Ex. Blood agar which distinguish types of hemolysis
4) Selective growth medium
-

4 - Contains compounds that prevents the growth of some bacteria while allowing the
prevent growth of other bacteria
'

allow
- Ex. Mannitol salt agar- Staphylococcus
- Dyes, sugar, high salt conc., antibiotics used to achieve selectively
Bacterial metabolism
Metabolism- All chemical and physical workings of a cell
1) Anabolism
biosynthesis
• enemy - Biosynthesis
require
faking
•

building
•
b
- Results in the synthesis of cell molecules and structures
Of
Synthesis
• molecule - Building and bond-making process
cell

- Require an input of energy

-

2) Catabolism
Degradative -
°
the Degradative
bonds
break
- Break the bonds of larger molecules into smaller ones
•

.pro#rgty
- Often produce energy
Carbohydrate metabolism
1) Fermentation
2) Respiration
- Aerobic respiration- total ATP-40-4-glycolysis
- 2-TCA
- 34-electron transport
- 6 CO2, 6O2, 6 water molecules
Microbial energy-yielding metabolism:
1) Glycolysis/Embden-Meyerhof-Parnas Pathway or substrate phosphorylation
- Occurs in the cytoplasm of all cells

=
- Does not require O2
2) Tricarboxylic cycle (TCA)/ Kreb’s cycle/ Critic acid cycle
- Occurs in the cytoplasm of prokaryotes and mitochondria of eukaryotes
3) Electron transport/ Oxidative phosphorylation
- Occurs in cell membrane of prokaryotes and mitochondria of eukaryotes
-

Fermentation
- An anaerobic respiration in which both the electron donor and final electron
acceptor are organic compounds
- Products
▪ Most prominent- alcohol beverages (wine, whiskey, bear)
▪ Solvents- acetone, butanol
▪ Organic acids- lactic, acetic
▪ Dairy products
1. Alcoholic fermentation
- Yeast species: acetobacter or gluconobacter (Vinegar prod.)
- Prod. Of beer and wine
- Products- Ethanol, CO2
2. Acidic fermentation
- Homolactic- product of fermentation is mainlyQ lactic acid
▪ Souring of milk
- Heterolactic- Mixture of lactic acid acetic acid & CO2
▪ Propionebacterium- propionic acid- Characteristics flavor to Swiss
cheese, CO2 produced holes
Enzymes
. proteinspeed - Proteins
catalyst recycled - Organic catalysts to speed up the rate of cellular reactions
- Can be recycled
: canbe - Ex. Penicillinase- Hydrolyzed beta-lactam reaction in substrate penicillin
 ✓
,

DISEASES
Role of microbial Enzymes in disease
- Many pathogens secrets isoenzymes that help them avoid host defenses or promote their
multiplication in tissues
- Contribute to pathogenicity- toxins or virulence factors
-- Strep. Pyogenes- streptokinase- dissolves blood clots & assists in invasion of wounds
-

- P. aeruginosa- Elastase and collagenase- digest elastin and collagen

-
-

-
- C. perfringens- Lecithinase C- damages cell wall and accts. For tissue death associated with
-

gangrene
Bacterial Death
- Senescence in stationary cultures, genetical- programmed or prophage- included cell
death, external noxious influences such as antibiotics or deliberate processes of
sterilization & disinfection
Sterilization- Inactivation of all self-propagating biological entities
- Renders a surface or product free from viable org.
Disinfection- reduction of pathogenic org. to a label which they no longer constitute a risk
Antisepsis- disinfection applied to a living tissue (wound)
-

Biocide- Chemical/physical agent which inactivates micro-organism

-

Bacteriostatic- Biocide that inhabits multiplication

Bactericidal- Biocide that = kills bacteria
Methods used in sterilization and Disinfection:
1. Heat- Only method of sterilization that is both reliable and widely applicable at
temperatures above 100 C
a. By moist heat
• 121 C for
15 mins • Hydrated proteins can be denatured with less energy
• Requires exposure at 121 C for 15 min
.

.nu#:i:.::n3roEEns • Autoclave temp> 100 C, pressure steam

• Steam is not toxic and non-corrosive; saturated & dry
b. By dry heat
• Destructive oxidation of essential cell components
• 160
C
for 2HRS
Destructive
OXIDATION • Killing of most resistant spores by dry heat req. temperature of 160 C for 2
tempt ) hrs
.

(higher
•

•
Incarnation heat
• Incineration- Efficient method of sterilization and disposal of contaminated
materials at a higher temperature
*
red
air • Red heat
* hot • Hot air sterilization
2. Ionizing radiation
- Beta & Gamma irradiation- For single use disposable items
- - -

- Reduces spoilage and remove pathogens

3. Filtration
- Removes bacteria and all larger micro-organism from liquids that are liable to be
-

spoiled by heating (blood serum, antibiotic solution)

- -

- Removes cysts of protozoa that are not killed by chlorination in the production of
-

drinking water
4. Gaseous chemical agents
- Ethylene oxide- Sterilization of plastics and other thermolabile mat. That cannot
-
-
withstand heating
-
5. Liquid chemical agents
- For heat-liable fiberoptic instruments (Flexible endoscopes)
-

The efficiency of particular method of chemical disinfection is dependent on the concentration and
stability of the agent, the no. type and accessibility of mico org., the temp, and pH, and the presence
of organic or other interfering subs.
In gen., gram (+) bacterial are more sensitive to disinfectants than Gram (-) bacteria; Microbacteria
and fungal spores are relatively resistant; bacterial spores are highly resistant
Disinfectants may be inactivated by hard tap water, cork, plastics, blood, urine, soaps and
detergents or another disinfectant
Choice of Method depends on:
 choice of method depends on:
1.) Nature of item to be treated
2.) Likely microbial contamination
3.) Risk of transmitting infection to patients or staff in contact with the item
- Choice is heat rather than chemicals
- All sterilizing and disinfecting agents have some action on human cells
Bacterial Defense against Noxious Chemicals:
1.) Preventing access-low cell envelope permeability
-

2.) Destruction the enzymes

-

3.) Lack if target

4.) Bypass of target
* lack

* bypass
 MicroBio: Bacterial Taxonomy
# study infectious
diseases
Taxonomy
• (Gk. taxon = arrangement; eg, the classification of organisms in an ordered system
that indicates a natural relationship).
• It is a vocabulary that consistently communicates the unique characteristics of
infectious organisms to students, microbiologists, and health care workers is critical
to avoid the chaos that would ensue without the organizational guidelines of bacterial
taxonomy
• Goal: accurately study infectious diseases

Criteria (way of naming an organism)

1. Structural
• Presence of pores: allows survival of microorganism in extreme environments
2. Physiologic
3. Biochemical
•
gram positive • F-Ferment specific carbohydrates y" (lactose);
• Gram staining (another biochemical means of identifying microorganisms),
• gram negative
divides bacteria into two major groups (gram positive, gram negative
microorganisms)
4. Genetic
• DNA-based technologies (Polymerase Chain Reaction ”PCR” reaction)
---------------------------------------------------------------

Identification, classification, and nomenclature are three separate but interrelated areas
of bacterial taxonomy. Each area is critical to the ultimate goal of accurately studying the
infectious diseases and precisely communicating these to others in the field.

*Identification is practical use of a classification scheme to:

1. Isolate and distinguish specific organisms among the mix of complex microbial flora,
2. Verify the authenticity or special properties of a culture in a clinical setting,
3. Isolate the causative agent of a disease.

The latter may lead to the selection of specific pharmacologic treatments directed toward
their eradication, a vaccine mitigating their pathology, or a public health measure (eg,
handwashing) that prevents further transmission.

Identification schemes are not classification schemes, although there may be some
superficial similarity. For example, the popular literature has reported Escherichia coli as
being a cause of hemolytic uremic syndrome (HUS) in infants. There are hundreds of
different strains that are classified as E coli but only a few that are associated with HUS.
These strains can be “identified” from the many other E coli strains by antibody reactivity
with their O-, H-, and K-antigens. However, they are more broadly classified as a member
of the family Enterobacteriaceae

*Classification is the categorization of organisms into taxonomic groups. Experimental and

-
-
-

observational techniques are required for taxonomic classification. This is because

biochemical, physiologic, genetic, and morphologic properties are historically necessary for
establishing a taxonomic rank. This area of microbiology is necessarily dynamic as the tools
continue to evolve (eg, new methods of microscopy, biochemical analysis, and
computational nucleic acid biology)

*Nomenclature refers to the naming of an organism by an established group of scientific

and medical professionals. This is arguably the most important component of taxonomy
because it allows medical professionals to communicate with each. Just as our societal
vocabulary evolves, so does the vocabulary of medical microbiology. Any professional
associated with infectious disease should be aware of the evolving taxonomy of infectious
microorganisms.
The taxonomic ranks form the basis for the organization of bacteria. Linnaean taxonomy
is the system most familiar to biologists.
 Formal Rank Example

Kingdom Prokaryotae

¥
Phylum/Division Gracilicutes

Class Scotobacteria

Order Eubacteriales

Family Enterobacteriaceae

= Genus

Sepies
Escherichia

Coli
✓
Subtype Escherichia coli O157:H7

The lower ranks are approved by a consensus of experts in the scientific community. Of
these ranks, the family, genus, and species are the most useful

---------------------------------------------------------------

CRITERIA FOR IDENTIFICATION OF BACTERIA

Growth on Media
• Suitable criteria for bacterial classification include many of the properties that were
described in the preceding chapter.
• One criterion is growth on different types of bacteriologic media.
• The general cultivation of most bacteria requires media rich in metabolic nutrients.
These media generally include agar, a carbon source, and an acid hydrolysate or
enzymatically degraded source of biologic material (eg, casein). Because of the
undefined composition of the latter, these types of media are referred to as complex
media.

Media can be nonselective or selective; the latter are used to distinguish among the various
bacteria in a clinical sample containing many different organisms.

• blood agar

chocolate agar
A. Nonselective Media •

Blood agar and chocolate agar are examples of complex, nonselective media, which
support the growth of many different bacteria. These media are intended to cultivate as
many species as possible, thus giving rise to numerous types of bacterial colonies.

B. Selective Media • inhibitory agent

reduce
- to eliminate a

• It is used to eliminate (or reduce) the large numbers of irrelevant bacteria in these
specimens because of the diversity of microorganisms that typically reside at some
sampling sites (eg, the skin, respiratory tract, intestines, vagina), selective media are.
• The basis for selective media is the incorporation of an inhibitory agent that specifically
selects against the growth of irrelevant bacteria.
• Examples of such agents are:

• Sodium azide only gham positive

-

• selects for gram-positive bacteria over gram-negative bacteria

• Bile salts (sodium deoxycholate)
• select for gram-negative enteric bacteria and inhibit gram-negative mucosal
and most gram-positive bacteria
• Colistin and nalidixic acid
of many negative bacteria
inhibits growth gram
-

-
B
 • inhibit the growth of many gram-negative bacteria

*Examples of selective media are MacConkey agar (contains bile) that selects for the
Enterobacteriaceae and CNA blood agar (contains colistin and nalidixic acid) that selects
for staphylococci and streptococci.

C. Differential Media

• Upon culture, some bacteria produce characteristic pigments; the activity of these
enzymes often can be detected as zones of clearing surrounding colonies grown in
the presence of insoluble substrates (eg, zones of hemolysis in agar medium
containing red blood cells).
• Many of the members of the Enterobacteriaceae can be differentiated on the basis
of their ability to metabolize lactose. For example, pathogenic salmonellae and
shigellae that do not ferment lactose on a MacConkey plate form white colonies,
• while lactose-fermenting members of the Enterobacteriaceae (eg, E coli) form red or
pink colonies. The number of differential media used in today’s clinical laboratories
is far beyond the scope of this chapter.

Microscopy -
is visualization by light microscopy

Historically, the Gram stain, together with visualization by light microscopy, has been
among the most informative methods for classifying the eubacteria. This staining technique
broadly divides bacteria on the basis of fundamental differences in the structure of their cell
walls. This typically represents the first step in identifying individual microbial specimens
(eg, are they gram negative or gram positive) grown in culture or even directly from patient
specimens (eg, urine specimens).

Biochemical Tests

1. Oxidase Test
• uses an artificial electron acceptor, can be used to distinguish organisms on the
basis of the presence or absence of a respiratory enzyme, cytochrome C, the
lack of which differentiates the Enterobacteriaceae from other gram-negative rods

2. Catalase Test
• catalase activity can be used, for example, to differentiate between the gram
positive cocci; the species staphylococci are catalase positive, whereas the
species streptococci are catalase negative.

3. Coagulase Test
• Staphylococcus aureus (coagulase positive)
• Staphylococcus epidermitidis (coagulase negative)
Immunologic Tests: Serotypes, Serogroups, and Serovars
# SUBTYPING
important , strains of given species
The designation “sero” simply indicates the use of antibodies (polyclonal or
-1$
•
a

to identify a particular stain

monoclonal) that react with specific bacterial cell surface structures such as
lipopolysaccharide (LPS), flagella, or capsular antigens.
• The terms “serotype,” “serogroups,” and “serovars” are, for all practical purposes,
identical—they all use the specificity of these antibodies to subdivide strains of a
particular bacterial species.
• Under certain circumstances (eg, an epidemic), it is important to distinguish among
strains of a given species or to identify a particular strain. This is called subtyping and
is accomplished by examining bacterial isolates for characteristics that allow
discrimination below the species level.

Genetic Diversity

Developments in molecular biology now make it possible to investigate the relatedness of

genes or genomes by comparing sequences among different bacteria

It should be noted that genetic instability can cause some traits to be highly variable within
a biologic group or even within a specific taxonomic group. For example, antibiotic
resistance genes or genes encoding enzymes (eg, lactose utilization) may be carried on
plasmids or bacteriophages, extrachromosomal genetic elements that may be transferred
among unrelated bacteria or that may be lost from a subset of bacterial strains identical in
all other respects.

Many organisms are difficult to cultivate, and in these instances, techniques that reveal
relatedness by measurement of nucleic acid hybridization or by DNA sequence analysis
may be of particular value.

---------------------------------------------------------------

CLASSIFICATION SYSTEMS

Dichotomous Keys
• Organize bacterial traits in a manner that permits logical identification of organisms.
The ideal system should contain the minimum number of features required for a
correct categorization. Groups are split into smaller subgroups based on the
presence (+) or absence (−) of a diagnostic character.

Numerical Taxonomy (Using Biochemical Measures of Activity)

NUMERICAL
*AXONOMY ( 1970 ) s

• Numerical taxonomy became widely used in the 1970s. These classification LB

, uses a huge number of unweighted ,

schemes use a large number of unweighted, taxonomically useful characteristics. taxonomically useful characteristics

For these assays, an individual bacterial colony must be isolated and used to )
iF⑧ individual bacterial colony (
isolated

inoculate the test format. One example of this is the Analytical Profile Index (APITM), PROFILE INDEX ( API TM)
MABEN ANALYTICAL
which uses numerical taxonomy to identify a wide range of medically important -
uses numerical taxonomy ,

microorganisms wide of medically important

range
• The limitation of this approach is that it is a static system. As such, it does not allow
microorganisms
for the evolution of bacteria and routine discovery of new bacterial pathogens
DB STATIC SYSTEM
for the evolution
Nucleic Acid–Based Taxonomy -
it does not allow

of bacteria a routine discovery of

new bacterial pathogens

• Since 1975, developments in nucleic acid isolation, amplification, and sequencing

spurred the evolution of nucleic acid–based subtyping systems.
• These include plasmid profile analysis, restriction fragment endonuclease
analysis, repetitive sequence analysis, ribotyping, and genomic sequencing.
Plasmid Analysis
 Daraa PIAS Mi DS
elements
dB extra chromosomal genetic
isolated )
✓ Isolated Bacterium (
✓ A grose Get Electrophoresis ( separated )
4¥ to determine NUMBER & SIZE

• Plasmids are extrachromosomal genetic elements. These can be isolated from an

isolated bacterium and separated by agarose gel electrophoresis to determine their
number and size.

---------------------------------------------------------------

DESCRIPTION OF THE MAJOR CATEGORIES AND GROUPS OF BACTERIA

BB EU BACTERIA
} replicate asexually
-
"

PROKARYOTIC ORGANISMS
*
-B AR CHAE BACTERIA

• There are two different groups of prokaryotic organisms, eubacteria and

archaebacteria. Both are small unicellular organisms that replicate asexually.

¥7
AEubacteria
BE no true nucleus • refer to classic bacteria as science has historically understood them.
to characteristics lipid • They lack a true nucleus, have characteristic lipids that make up their membranes,
BB
✓ PEPTDOGY
CAN possess a peptidoglycan cell wall, and have a protein and nucleic acid synthesis
CELL WALL
inhibited machinery that can be selectively inhibited by antimicrobial agents.
• be
selectively
antimicrobial agents
Archaebacteria
* • do not have a classic peptidoglycan cell wall and have many characteristics (eg,
⑧ NO classic peptidoglycan
protein synthesis and nucleic acid replication machinery) that are similar to those of
characteristics
cell eukaryotic cells
HB have many
Dun similarto those Eukaryotic
is protein synthesis A nucleic acid replication
---------------------------------------------------------------

The Eubacteria

A. Gram-Negative Eubacteria
complex • This is a heterogeneous group of bacteria that have a complex (gram-negative type) cell
Heterogeneous group ,
⑨

B
' r L ) gram negative
-
-
envelope consisting of an outer membrane, a periplasmic space containing a thin
"

BINARY FISSION "

peptidoglycan layer, and a cytoplasmic membrane.
B.
Reproduction BUDDING
-
produce
someby • The cell shape may be spherical, oval, straight or curved rods, helical, or filamentous;
be phototropic
phototropic some of these forms may be sheathed or encapsulated.
or non

may
-

• Reproduction is by binary fission, but some groups reproduce by budding. Fruiting

BB MOTILITY
occurs by means
of FLAGELLA bodies and myxospores may be formed by the myxobacteria.
• Motility, if present, occurs by means of flagella or by gliding motility.
-

GLIDING MOTILITY
by or

• Members of this category may be phototrophic or nonphototrophic bacteria and include

aerobic, anaerobic, facultatively anaerobic, and microaerophilic species

B. Gram-Positive Eubacteria
wall
DB has a cell
• These bacteria have a cell wall profile of the gram-positive type; cells generally, but not
DA ( *) gram positive .

always, stain gram positive.

-
thick cell wall • The cell envelope of gram-positive organisms consists of a thick cell wall that determines
shape A
determine cellular
cellular shape and a cytoplasmic membrane.
-

membrane
cytoplasmic • These cells may be encapsulated and can exhibit flagellar-mediated motility.
FISSION
Reproduction :! BINARY
• Cells shape may be spherical, rods, or filaments; the rods and filaments may be
pumps

nonbranching or may show true branching.

• Reproduction is generally by binary fission.
• Some bacteria in this category produce spores (eg, Bacillus and Clostridium spp.) as
resting forms that are highly resistant to disinfection.
• The gram-positive eubacteria are generally chemosynthetic heterotrophs (see Chapter
5) and include aerobic, anaerobic, and facultatively anaerobic species.
• The groups within this category include simple asporogenous and sporogenous bacteria
as well as the structurally complex actinomycetes and their relatives.

C. Eubacteria Lacking Cell Walls

• These are microorganisms that lack cell walls (commonly called mycoplasmas and
making up the class Mollicutes) and do not synthesize the precursors of peptidoglycan.
• They are enclosed by a unit membrane, the plasma membrane.
• They resemble the L-forms that can be generated from many species of bacteria (notably
gram-positive eubacteria); unlike L-forms, however, mycoplasmas never revert to the
walled state, and there are no antigenic relationships between mycoplasmas and
eubacterial L-forms.
• Six genera have been designated as mycoplasmas on the basis of their habitat;
however, only two genera contain animal pathogens.
• Mycoplasmas are highly pleomorphic organisms and range in size from vesicle-like
forms to very small (0.2 μm), filterable forms (meaning that they are too small to be
captured on filters that routinely trap most bacteria).
• Reproduction may be by budding, fragmentation, or binary fission, singly or in
combination.
• Most species require a complex medium for growth and tend to form characteristic “fried
egg” colonies on a solid medium.
• A unique characteristic of the Mollicutes is that some genera require cholesterol for
growth; unesterified cholesterol is a unique component of the membranes of both sterol-
requiring and non–sterolrequiring species if present in the medium.