1.

4 Basic Concepts in Microbiology MICROBIOLOGY

Dr. Eduardo| June 25, 2013

OUTLINE
I. Bacterial Classification, Nomenclature and Identification
A. Classification
B. Nomenclature
C. Identification
II. Bacterial Cell Structures
A. Eukaryotes, Prokaryotes and Archaebacteria
B. Structures of Gram (+) and Gram (-) Bacteria
MICROBIOLOGY
C. External Structures of Prokaryotes
D. Internal Structures of Prokaryotes
III. Microbial Growth
A. Factors that Affect Microbial Growth
B. Oxygen Requirements of Organisms
C. Bacterial Growth
D. Phases of Growth Curve
IV. Methods of Controlling Microbial Growth
A. Important Terms in Controlling Microbial Growth
B. Actions of Microbial Agents
C. Methods of Evaluating a Disinfectant
V. Normal Flora/Microbiota

I. BACTERIAL CLASSIFICATION, NOMENCLATURE, AND

IDENTIFICATION Figure 1. Taxonomic Ranks
A. Classification
 categorization of organisms into taxonomic groups/ranks  Taxonomic ranks form the basis for organization of bacteria
e.g FAMILY GENUS SPECIES  Species in prokaryotic-individual strains sharing high degree of
Enterobacteriaceae Escherichia coli similarity in many features
 requires experimental and observational techniques to adequately  Methods Used in Classifying Bacteria
described a certain taxon or organism depending on the following 1. Intuitive method (subjective)
properties: o by expert microbiologist
1. Biochemical - e.g Oxidase (-), IMViC ++- - o by familiarization of the properties of the organism studied for
IMViC – Indole, Methyl Red, Voges-Proskauer, “in” Citrate several years
– a set of biochemical tests to aid in the identification of 2. Numerical taxonomy (objective)
coliforms o by determining many characteristics (around 100-200) for each
2. Physiological – facultative anaerobe, ferments lactose strain studied
3. Morphologic – Gm (-) rod, motile o giving each characteristics equal weight
4. Genetic – with the development of molecular biology it is now o use of a computer to calculate % similarity (%s)
easy to investigate the relatedness of the genes or genomes by %s = __NS__
comparing their sequence with other bacteria NS +ND
where:
NS: # of characteristics same in the 2 strains
B. Nomenclature
ND: # of different characteristics
 naming of organisms by International rules that are established by
recognized group of medical professionals according to its:
 strains with a high % s to each other are placed into
one group
o characteristics – Hemophilus influenza (“Heme”-blood, “Philus”-
love, discovered during a pandemic of flu)
3. Genetic Relatedness (most reliable)
o discoverer – Salmonella shiga (Dr. Shiga isolated the organism)
o based on the degree of relatedness between organisms on
o persons affected – Legionella pneumophila (affected the
their hereditary material (DNA)
American Legion, suffered pneumonia)
o 2 basic principles :
o named after a geographical place – Gallaecimonas
a. DNA homology
pentaromativorans
b. Ribosomal RNA homology
o after a person that is not necessarily associated with it –
o rRNA oligonucleutide cataloging – modern complex technique
Bartonella sp.
o rRNA – coded by rRNA cistrons
 Taxonomist – uses DNA sequencing to determine their relatedness
o rRNA cistrons – fraction of DNA molecules
(pure culture must be achieved)
o Even if 2 organisms are only distantly related and show no
significant DNA homology there still maybe considerable
C. Identification similarities in the nucleotide sequence of the rRNA cistrons
 refers to a practical use of the classification scheme for: o Cistron/Intron/Exon – structural gene of either DNA or RNA
1. Isolation
2. Verification

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Group 13: Gilera, Goco, Gopez, Gonzales, Guillermo, Guingon, Gumiran, Hernandez, H., Hernandez, M.
 MICROBIOLOGY 1.4
 By International agreement, each species has only one Officially 2. Gram reaction
Accepted Name o Positive – take up crystal violet dye making it bluish purple
o E.g. Escherichia coli is called Coprobacterium intestinale in other o Negative – take up the crystal violet dye but it is decolorized
countries with alcohol and take up the secondary dye (safranin red)
o Important terms: instead, making it red in color
 Strain – descendant of a pure culture 3. Oxygen requirement
 Species – collection of strains having similar characteristics o Obligate anerobe – no O2 requirement
 Genus – collection of similar species o Facultative anaerobe – aerobe/anaerobe
 E.g. GENUS SPECIES STRAIN o Microphiles – reduced O2requirement
Staphylococcus aureus ATCC 1897 o Obligate aerobe – requires O2
o Chosen characteristics must be combined and must occur only in 4. Presence of spores – Clostridium sp., Bacillus anthracis
that particular kind of organism and no other organisms 5. Biochemical Tests
 E.g. E. coli – using battery of biochemical test o Sulfide-Indole-Motility test – makes use of waste products and
Indole (+) ,MR (+) VP( -,) Citrate( -) enzymes of bacteria to identify them
st
 Bergey’s Manual of Systematic Bacteriology (1923, 1 ed.)– used to  Blackening: H2S; Pink: uses tryptophan producing indole;
classify bacteria according to phylogenetic relationship, included motility: growth
30,000 species o Serology typing
 International Commission on Systematic Bacteriology (1980) – 6. Media Growth Tests
published the approved list of bacterial names (2,500 species) o Types of nutrient requirements for growth in media - Blood
agar plate for RBC requiring bacteria such as Heamophilus
influenza
o Colony Size and Characteristic – small, pin-sized, large, mucoid
o Hemolysis test – Lancefield classification of hemolysis
7. Presence of virulence factors/Pathogenicity - quantitative ability
of the oraganism to cause disease
o Presence of capsule
o Presence of toxins such as hemolyzin
o Presence of certain enzymes – catalase which reacts with H2O2,
creating oxygen
8. Genetic classification

II. BACTERIAL CELL STRUCTURES

A. Eukaryotes, Prokaryotes, and Archaebacteria
 Eukaryote – true nucleus, nuclear material encased in nucleolus
 Prokaryotes – bacteria, does not have a nucleus but possesses a
nuclear region
o Archaebacteria – ancient bacteria, does not have a peptidoglycan
cell wall
o Eubacteria – true bacteria, includes human pathogens
 Characteristics
o Size: 0.2 – 2.0 um diameter, 2 - 8 um in length
o Shape
 Cocci: diplo, strep, clusters
 Bacilli : diplo, chain, coccobacilli
Figure 2. Criteria for Bacterial Classification See appendix for Images of Criteria for
Classification
 Spiral: curved, corkscrew, helical
 Mostly contains single chromosome (approximately 10,000
 Classification genes)
1. Shapes o Nucleoid – contains the DNA
o Cocci – round, oval shape
 Diplococci – kidney-shaped, coffee bean-shaped e.g. B. Structure of Gram (+) and Gram (-) Bacteria
Neisseria gonorrheae
 Streptococci – in chain e.g. Streptococcus pneumoniae
 Staphylococci – grapelike cluster formation e.g.
Staphylococcus aureus
o Bacilli – rod-shaped, single
 Coccobacilli – short with rounded ends e.g. Escherichia coli
 Streptobacilli – in chains e.g. Bacillus anthracis
o Spirillum – spiral
 Vibrio - comma shaped e.g. Vibrio cholera
 Spirillum - helical, like cork screw with rigid bodies e.g.
Leptospira interrogans
 Spirochete – helical but flexible e.g. Treponema pallidum
Figure 3. Structures of Gram(+) and Gram(-) Bacteria
Coccobacilli
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 MICROBIOLOGY 1.4
2. Axial Filaments – for motility
 Spirochetes – e.g. Treponema pallidum (syphilis), Leptospira spp

Figure 4. Difference between the properties of Gram Positive and Gram Negative Cell Walls

Figure 6. Axial Filaments (red thread-like on the left figure)

 Gram (+)Bacterial Cell Wall – components function in porosity and
elasticity properties of the envelope 3. Flagella – made of protein (highly antigenic), e.g. “H” Ag
o Thick peptidoglycan (20-80nm) (Salmonella)
o Teichoic acid
 Monotrichous - single polar flagellum
 Wall Teichoic Acid (WTA) - linked to peptidoglycan
 Amphitrichous - one flagellum on both ends
 Lipoteichoic Acid (LTA)
 Lophotrichous - multiple polar flagella
 Gram (-)Bacterial Cell Wall
 Peritrichous - flagella distributed over the entire cell
o Components
 Thin peptidoglycan (5-10nm)
 Lipopolysaccharide( LPS )
 Lipoprotein (hydrophilic)
o Outer Membrane – contains “porins” that permit the passive
diffusion of low molecular weight hydrophilic compounds like
sugar and amino acids
 Permeability varies from species to species, e.g. Pseudomonas
aeruginosa is 100x more permeable than E.coli

C. External Structure of Prokaryotes

1. Glycocalyx
 Functions Figure 7. Types of Flagella
o Anti phagocytosis
o Avoidance of the killing effect of lysosome enzymes 4. Fimbriae/Pili
o Paralyze leukocyte  Attachment to other bacteria and other membranous surfaces
o Protect anaerobic cells from O2 toxicity (e.g. capsule of B. (Host cells)
fragilis)  Provide site for main attachment of bacteriophage (bacterial
 Types: viruses)
o Capsule (some are antigenic )  Pili of different bacteria are antigenically distinct and elicit the
 Polysaccharide in nature formation of antibodies
 Firmly attached to the cell wall (e.g. H. influenza type b)  Ab against the pili of one bacteria will not prevent the attachment
 Induce Ab production against “ K Ag “ of another species
 Use for vaccine production  Types of Pili
o Slime Layer o Ordinary Pili – for adherence
 Loosely attached to the bacterial cell o Sex Pili – in gene transfer during conjugation
 Used for attachments to rocks, teeth and plastic tubing (e.g.  Category of Fimbriae:
Pseudomonas aeruginosa, Strep mutans, Rikettsiae) o Adhesins (attachment) – host tissue cell specific
 Examples: K99 (fimbriae of E. coli), Fimbriated Neisseria
gonorrhea (type 1, type 2), M. protein (Strep pyogenes)
virulence factor
o Evasins – evade being phagocytized
 M-protein (Strep. pyogenes)
o Aggresins – destroys leukocytes
 M. protein

5. Spores and Endospores – Genus Bacillus; Clostridium

 Sporulation – process of forming spores as means of survival
when their moisture & nutrient supply is low
o Genetic materials are enclosed in several proteins coats that
are resistant to heat, drying and most chemical
Figure 5. (a) Rickettsia; (b) Rickettsias in chicken embryo cell
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o Germinates back into vegetative form in moist and nutrient
rich surface
Figure 8. Endospore Formation
6. Cell wall – Responsible for the shape
 Prevent cells from rupturing when the water pressure inside the
cell is greater than the pressure outside
 Composed of peptidoglycan (murein)
 Determines the ability to some species to cause disease (LPS with
Endotoxin of gram (-) rods
 Site of action of some antibiotics (e.g. penicillin)
 Use to differentiate major type of bacteria
 Three complex polymer:
o N-acetylglusamine and N-acetylmuramic acid – backbone,
same in all bacteria species
o Tetrapeptide side chain attached to N acetylmuramic acid
o Identical peptide cross bridges species – varying from species
to species, same with the side chain
 Endotoxin – found in Gram (-) cell wall
o Induces fever, hemorrhagic necrosis, disseminated
intravascular coagulation
o E.g. typhoid fever – production of tumor necrosis and
activation of alternate complement pathway
 Enzymes that can attack cell walls of prokaryotes:
o Lysozyme (saliva, tears, nasal secretions) hydrolysed the
backbone of peptidoglycan
o Enzymes withAutolysin (e.g. amidases, peptidases) possess by
the bacteria themselves during (plays an important role during
autolysis - cell death)
 Bacteria without cell walls – without cell walls, atypical or very
little cell wall materials, smallest, can reproduce outside living
host cells
MICROBIOLOGY 1.4
Figure 9. Removal of Bacterial Cell Wall Methods
D. Internal Structure of Prokaryotes
Figure 10. Structures inside Prokaryotic Cell
Cell Membrane Functions:
 Selective permeability , transport of solutes
 Electron transport & oxidative phosphorylation in aerobic species
 Excretion of hydrolytic exoenzymes
 Biosynthesis of DNA, cell wall polymers, membrane lipids
1. Cytoplasm – substance of cell inside the plasma membrane
 80% water
 Includes proteins (enzymes), carbohydrates, lipids
 Without sterols
2. Cytoplasmic Particles
 Ribosomes - sites of protein synthesis, 70S ribosomes
 Inclusion Bodies (stores energy, e.g. glycogen – source of carbon)
 Granules - source of inorganic phosphates, found in some
bacteria (e.g. C.diphtheriae)
3. Nuclear Area/Nucleoid
 Mostly contains single-long continuous circularly arranged thread
of ds – DNA (haploid chromosome)
 # of chromosomes depends on size and speed of growth
Note: Check Appendix for Tables on Bacterial Structures.
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 MICROBIOLOGY 1.4

II. MICROBIAL GROWTH  Most bacteria grow between pH 6.5 and 7.5
 Molds and yeasts grow between pH 5 and 6
Orderly increase of all chemical constituents of cell  Acidophiles: pH 3

 Entails the replication of all cellular structures, organelles  Alkaliphiles: pH 10.5
and protoplasmic components from the nutrients present in  Neutralophiles: pH 6-8
the surrounding environment.
 Increase in cell size
 Increase in cell number

A. Factors that Affect Bacterial Growth

1. Availability of nutrients
 Energy needed to synthesize cellular components
o Iron – pathogenic bacteria use intracellular sidiphores to
harvest iron from the host
o Carbon – autotrophs
 require only water, inorganic salts and CO2
 Synthesize essential organic metabolites from CO2
4+
o Nitrogen – NH is the source of the nitrogen atom of amino
acid, purines, pyrimidines and other biomolecules
o Protein
o Sugars
 Growth Factor Figure 11. Effect of temperature on bacterial growth
o Prototrophic – synthesizes their own growth factor
o Autotrophic – needs exogenous source of growth factors, i.e. B 5. Osmotic pressure and salinity
complex vitamins, amino acids, purines and pyrimidines  Osmotic pressure – pressure that is exerted on a cell membrane
by solutions both inside and outside the cell
Table 1. Classification of bacteria based on CO2 source.
 Salinity – concentration of osmotically active solutes inside a
Classification of Bacteria Carbon Source Energy source bacterial cell is in general, higher than the concentration outside
Phototrophs CO2 Light the cell.
Chemolitotrophs CO2 Chemical reaction  Majority of bacteria are unusually osmotically tolerant except for
Photoorganotrophs Organic material Light the mycoplasma and other cell- wall defective microorganism.
Chemoorganotrophs Organic material Chemical reaction  Membrane derived oligosaccharides (MDO) is the principal
source of fixed anion in the periplasmic space and acts to
2. Moisture/Water maintain the higher osmotic pressure and Donnan membrane
 All microbes require water for growth potential of the periplasmic space.
 Cells consists of 70-95% water  Halophilic bacteria – salt loving
 Endospores and cysts can survive complete drying process.  Haloduric bacteria – tolerant to high concentration of salt

3. Temperature
 3 parameters
o Minimum growth temperature
o Optimal growth temperature
o Maximum growth temperature
 Bacteria have a upper and lower temperature within which it
grows
 Optimal temperature differ from each bacterium
o Thermophiles – 60 °C
 Minimum temp – 40 °C
 Maximum temp – 75 °C
o Mesophiles – normal body temperature
o Psychrophiles
o Psychotrophs – Refrigerator temperature
o Psychroduric organism – prefers warm, however they can
endure freezing temperature (e.g. E. coli, Salmonella typhi) but
not multiply

4. pH – alkalinity or acidity
 Bacterial species survive within a range of pH values but there is
also an optimal pH.

 Most microorganisms prefer a neutral or slightly alkaline medium
Figure 12. Effect of Osmotic Pressure on Bacteria
(pH 7-7.4)
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 MICROBIOLOGY 1.4

B. Oxygen Requirements of Organisms

Figure 14. Effect of O2 on the growth of vaious types of bacteria

C. Bacterial Growth
Figure 13. Oxygen requirements of organisms  Orderly increase in all constituents of cells
 Refers to the number of cells , not the size of cells
• Bacteria, but need catalase and superoxide dismutase to utilize  Bacteria reproduce by binary fission. They undergo exponential of
oxygen since the use of oxygen generates 2 toxic molecules: logarithmic growth
hydrogen peroxide (H2O2) and the radical superoxide (O2)
o Catalase: 2H2O2 →2H2O + O2
+
o Superoxide Dismutase (SOD) :2O2 + 2H → H2O2 + O2
• Possible targets for damage by H2O2 and O2 includes:
o Specific outer membrane proteins
o Redox active components of the cytoplasmic membrane
o Enzymes in periplasmic space

1. Obligate aerobes
o Requires oxygen for growth because their ATP- generating system
is dependent on oxygen as the hydrogen acceptor
o Catalase and superoxide dismutase neutralize toxic forms of
oxygen
2. Facultative anaerobes
o Capable of growth under both aerobic and anaerobic conditions.
However greater growth in the presence of oxygen
o Utilize oxygen to generate energy by respiration if it is present,
but they can use the fermentation pathway to synthesize ATP in Figure 15. Bacterial Population Growth curve
the absence of sufficient oxygen (E.g. E. coli)
3. Obligate anerobes  Generation Time (GT) – the time it takes for one cell to become two
o Cannot tolerate oxygen due to the absence of catalase and cells by binary fission.
superoxide dismutase. o Higher GT: slower growth
4. Aerotolerant anaerobes o Lower GT: faster growth
o Anaerobic growth but can tolerate the presence of oxygen due to Ex. E. coli, V. cholerae, Staph., Streptococcus - 20 mins.
the presence of SOD Pseudomonas and Clostridium - 10 mins.
5. Microaerophiles M. tuberculosis - 18 to 24 hours
o Requires oxygen in low concentration  Bacterial population growth curve – determined by growing a pure
o Exposure to normal atmospheric oxygen is toxic culture the organism in a liquid medium at constant temperature.
Data are plotted on a graphic paper plotting the logarithm (log 10)
of the number of viable bacteria (y-axis) against the incubation time
(x-axis)

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 MICROBIOLOGY 1.4

III. METHODS OF CONTROLLING MICROBIAL GROWTH

 Sterilization – complete destruction of all living organism including
cells, viable spores, viruses
E.g. Dry heat

Autoclaving (steam under pressure)
Gas (ex. Ethylene glycol)

Various chemicals (formaldehyde)
Radiation (UV, gamma rays)

A. Important Terms in Controlling Microbial Growth

 Disinfection – removal of pathogens from nonliving objects by
physical or chemical methods
 Disinfectants – are strong chemical substances use to disinfect
inanimate objects
 Antiseptic – solution used to disinfect skin or other living tissues
(use of alcohol)
Figure 15. Bacterial growth in a pure culture
 Sanitation – reduces microbial population to a level considered safe
by public health standards such as those applied to restaurants
 Microbiostatic Agent – drug or chemical that inhibits the growth
D. Phases of Growth Curve
and reproduction of microorganism

1. LAG phase – NO MULTIPLICATION E.g. Concentrated sugar & salt solution – will lyse the RBC
 Bacteria absorb nutriients, synthesize enzymes and prepare for Antimicrobial agents
cell division Liquid Nitrogen

 No cell division yet  Bacteriostatic agents – one that specifically inhibits the metabolism
2. LOG phase – doubles with each GT and reproduction of bacteria. They don’t multiply.

 Divides at maximal rate  Lypophilization – process combining dehydration and freezing used:
 Number of organisms double with each generation time o To preserve foods, such as fresh or cooked food is 
passed
 Bacterial population is most uniform in terms of 
chemical and through a vacuum chamber where 98% of moisture is extracted.
physiologic properties (culture used in biochemical and The moisture-free food is sealed in a moisture/O2 free package.
physiologic studies) When it is opened water is replaced regaining its flavor aroma.
3. Stationary phase – Growth = Death o Antibiotics (powder form placed in capsules)
 Greatest population density o Anti-sera
 Attained by bacteria at a population level of around 109 cells/mL o Microorganisms (for culture and recovery of organism)
 Total number of viable microorganisms remain 
constant due to: VIII. Pasteurization – heating until pathogens are destroyed
o Nutrient limitation
o Limited O2 availability B. Actions of Microbial Agents
o Accumulation of toxic waste products
o Change in pH  Alternation of membrane permeability
4. Death phase – Death>Growth  Damage to proteins
 Culture may die completely  Damage to nucleic acids
 Death is usually logarithmic.
 Because of total depletion of nutrients and I. Physical Methods to Inhibit Microbial Growth
a. Heat – microorganisms die when exposed
 Building up of toxic waste o Thermal Death Point (TDP) - lowest temperature at which all
cells in a culture are killed in 10 min. (Tap water is
recommended to boiled for 10 mins to kill the microorganisms)
o Thermal Death Time (TDT) - time to kill all
o cells in a culture
o Decimal Reduction Time (DRT) - minutes to kill
o 90% of population at a given temperature
o Dry Heat – oven, 160 to 165C for 2 hours or 170 to 180 C for 1
hour
o Incineration – burning of contaminated disposable materials
o Moist Heat – denatures proteins
b. Autoclave – large pressure cooker, Steam under pressure; 15psi,
121.5C, 20 minutes
c. Cold – slowing down of cell metabolic activity
o Low temperature inhibits microbial growth
o Refrigeration
o Slow freezing
o Rapid freezing (eg. Liquid N)
Figure 16. Bacterial population growth curve, with phases o Lypophilization (freeze drying)
d. Desiccation – absorbs moisture and prevents metabolism
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 MICROBIOLOGY 1.4
e. Radiation - Damages DNA IV. NORMAL FLORA/MICROBIOTA
f. Ionizing radiation - High energy radiation capable of producing  Collection of species routinely found in healthy, normal individuals
ionization in substance through which it passes, e.g., X-rays,
gamma rays, electron beams
A. Distribution of Normal Flora
g. Non-ionizing radiation (ultraviolet rays) – ultrasonic waves
Microwaves – kill by heat; not especially antimicrobial
h. Filtration - procedure undergone by drinking refill stations

II. Use of Chemical Agents to Inhibit Microbial Growth

Chemical disinfection – refers to the use of chemical agents
to inhibit the growth of pathogens. Either temporary or
permanent

How chemical disinfectant kills microorganisms?

1. Target and destroy cell membranes
E.g. Alcohol, chlorhexidine, triclosan
Handwash soap – triclosan
Liquid bleach – contains 10% chlorine
Listerine – alcohol and benzoic acid
Oracare – chlorine dioxide
2. Destroy enzyme and structural enzymes
3. Hydrogen peroxides, salt of heavy metals, formaldehyde and
ethylene oxide Figure 19. Normal bacterial flora in humans
4. Attack cell wall or nucleic acids
Distribution depends on:
C. Methods of Evaluating a Disinfectant  Amount of moisture present
3 4 2
Disk Diffusion Method o Dry area: 10 -10 organisms/cm
6 7 2
o Wet area: 10 -10 organisms/cm
 pH
 Temperature
 Salinity
 Chemical waste (urea, fatty acids)
 Presence of other microbes
 Washing decreases 90% of cell count

Table 2. Normal Flora in the different parts of the body

Figure 17. Filter paper with chlorine wherein Staphylococcus aureus and E. coli are Oral Strep viridans Large E. coli
sensitive. Pseudomonas aeruginosa is resistant.
Cavity Fusobacterium Intestines Ptoteus
Actinomycetes Klebsiella
Candida Lactobacilli
Candida
Pseudomonas
Bacteriodes
Clostridium
Enterococci
Nose Staph. Aureus Urethra Strep spp
Staph. Epidermidis Mycobacterium
Corynebacterium spp
E. coli
Bacteriodes
Throat Strep. spp. Vagina Lactobacillus
Neisseria meningitidis Strep grp B
Corynebacterium Candida
Haemophylus spp Gardnerella
Mycoplasma Vaginalis
Skin S. Epidermidis
Propionebacterium
acness
Pityrosporum ovale

Figure 18. Resistance of different bacteria

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 MICROBIOLOGY 1.4
Normal Flora per System
I. SKIN
 The high moisture (axilla, groin, and areas between the toes)
supports the growth of bacterial cells
 These are generally nonpathogenic and considered to be
commensal
 Cocci
o Staph epidermidis (90%)
o Staph aureus
 Yeast
o Candida albicans – scalp
o Pityrosporum ovale – stratum corneum and hair follicles
o Pityrosporum orbiculare – also stratum corneum and hair follicles
 Gram (-) rod Figure 22. Anaerobic flora in Upper respiratory system
 Pseudomonas aeruginosa – moist area
III. CONJUNCTIVA
 Blinking mechanically washes away foreign objects including
bacteria. Tears contain bactericidal lysozyme
 Staphylococcus epidermidis and Propionibacterium acnes are
dominant. Staphylococcus aureus, some streptococci,
Haemophilus sp. and Neisseria sp. are occasionally found.

IV, ORAL CAVITY

 Oral bacteria include streptococci, lactobacilli, staphylococci and
corynebacteria, with a great number of anaerobes, especially
bacteroides.
 The eruption of the teeth during the first year leads to
Figure 19. Propionebacterium acne (anaerobic) – puberty
colonization by S. mutans and S. sanguis.
 The oral flora :
II. RESPIRATORY TRACT o Usual cause of various oral diseases in humans, including
abscesses, and periodontal disease.
Upper Respiratory o Usually contain mixtures of bacteria with Bacteroides
melaninogenicus often playing a dominant role

Table 3: Normal Flora in the Oral Cavity

Aerobic
Strep mutans Neisseria meningitidis
Strep pyogenes Lactobacilli
Strep pneumonia Candida
Staph Haemophilus influenza
Anaerobic
Fusobacterium Found in gingival
Bacteroides crevices

Note: Mouth microbial density: 1011 per gram wet wt. of tissue
Figure 20. Aerobic flora in the Upper Respiratory System (mucous membrane)

V. GASTROINTESTINAL TRACT
Upper GI tract of Adult Humans
 Esophagus
o Contains only the bacteria swallowed with saliva and food
 Stomach (pH 1.5)
o Harbors transient flora Helicobacter pylori (gram -, urease +)
 Small Intestine (104 organism/g)
o Enterobacter – E. coli
o Candida albicans
o Lactobacillus
 Large intestine (1011 organisms/g)
o Predominant species are anaerobic Bacteroides, Clostridium
and anaerobic lactic acid bacteria in the genus Bifidobacterium
Figure 21. Aerobic flora in the Upper Respiratory System bifidum.

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 MICROBIOLOGY 1.4

Figure 23. Normal flora in GIT

Figure 26. Disturbance of Ecosystem in the vagina

Figure 24. Example of Normal Flora in GIT

Intestinal Tract of the Newborn

 Breast fed infants:
o Soft, light yellow – brown stool Figure 27. Body defenses in different areas
o Lactobacillus bifidus
 Artificially fed infants: TRANSIENT MICROFLORA/TEMPORARY RESIDENT FLORA
o Hard, dark brown, foul smelling stool  Why temporary?
o Lactobacillus acidophilus o Washed by bathing
o Cannot compete with the resident flora
VI. UROGENITAL TRACT o May fail to survive an acidic or alkaline environment
 Pathogens: o Flushed out (urine, feces, tears, perspiration)
o Lactobacillus, Group B Strep (Strep agalactae), Candida E.g. GIT – Clostridium tetani (gram negative rod with spore)
albicans, Staph epidermidis, Enterococcus faecalis
ROLES OF INDIGENOUS MICROFLORA
 Source of nutrient (Vit. K, Vit B12, pathothemic acid, pyridoxine,
biotin – from coliform bacteria)
 Act as antigens to stimulate the immune system
 Do not allow other microbes in establishing themselves to
produce infection by:
o competing for food,
o controlled pH,
o maintain O2 level,
o produce antibiotic-like subs that destroys other organisms (e.g
colicin of E. coli)

Figure 25. Normal flora in the vagina

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Figure 28. Role of Normal Flora in preventing disease production
Figure 29. Normal Flora blocking the pathogen
Figure 30. Normal Flora becoming opportunistic pathogen
MICROBIOLOGY 1.4
SUMMARY
1. Bacteria occur as spheres (cocci), rods (bacilli) or spirals.
2. All bacteria are classified as Gram-positive (retain the gram stain)
or Gram-negative (do not retain the gram stain).
3. Structural features of bacteria that are not seen in the human
cell, or differ from those in the human cell, include a capsule, an
outer membrane, a periplasmic space, a rigid cell wall, a
cytoplasmic membrane lacking sterols, the mesosome, flagellum,
fibrae (pili), 70S ribosomes, endospore, lack of a nuclear
membrane, plasmids and a haploid chromosome.
4. The major antigens of the bacterial cell are the capsule (K-
antigen), the lipopolysaccharide (O-antigen) and the flagellum (H-
antigen).
5. The growth cycle of a culture of bacteria is divided into four
phases: lag phase, exponential phase, stationary phase, decline
phase.
6. The capsule of bacteria are most commonly polysaccharide in
nature but proteinaceous in at least two species, Bacillus
anthracis and Yersinea pestis.
7. The capsule is a major virulence factor that allows bacteria to
evade phagocytosis, avoid the killing effects of lysosomal
enzymes, avoid complement-mediated cell lysis, paralyze
leukocytes, induce pathology in the host tissue, adhere to the
host cell, protect anaerobic cells from oxygen toxicity, produce a
unique colony type, enhance its pathogenicity, adsorb
bacteriophage and induce antibody synthesis.
8. Bacteria with capsules from smooth (S) colonies; those without a
capsule from rough (R) colonies; those with hydrophilic capsules
from mucoid (M) colonies
9. Serologically, the capsule is important in diagnosis, vaccine
production and as the basis for the Quellung reaction.
10. The cell wall of bacteria is made up sheets of cross-linked
repeating units of peptidoglycan. In Gram-positive cells this is
relatively thick as compared to Gram-negative cells.
11. Linked to the cell wall of bacteria are teichoic acids, cell wall
specific polysaccharides and, in some cases, proteins of special
significance.
12. Gram-negative bacterial cells contain lipopolysaccharide (LPS) in
their outer membrane. This is the source of the O-antigen and
endotoxin.
13. The functions/effects of the cell wall include maintenance of the
morphology or the bacterial cell, action as an adjuvant, induction
of fever, induction of sleep, competition with serotonin for
receptors on macrophages, induction of inflammation, induction
of liver granuloma, stimulation of hemopoietic stem cells,
induction of bowel inflammation, induction of antibody
synthesis.
14. Endotoxin induces fever, hemorrhagic necrosis (Shwartzman
reaction), disseminated intravascular coagulation, production of
tumor necrosis factor, activation of the alternate complement
pathway, stimulation of bone marrow cell proliferation,
enhancement of the immune and the Limulus lysate reaction.
15. The lipoteichoic acid of Gram-positive bacteria acts similar to the
endotoxin of Gram-negative bacteria.
16. Pili contain adhesins which allow the bacterial cell to bind to
human cells.
17. Flagella are organs of locomotion that are used in serotyping
strains of bacteria.
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APPENDIX

Morphology, Arrangement, and Gram Reaction. a. Types of bacterial shapes (top.) b. Gram positive cocci in chain e.g. Streptococcus pyogenes
(bottom left) c. Gram negative diplo-cocci located intracellulary (bottom middle) d. Gram positive cocci in pairs (bottom right)

Atmospheric requirements ( e.g. O2, CO2). From left to right, Toxin (hemolyzin) production. a. Gamma hemolysis (no hemolysis)
M. tuberculosis, H. pylori, E. coli, Clostridium tetani b. Beta hemolysis (complete clearing) c. Alpha hemolysis (green)

Table 1. External Structures of a Bacterial Cell

Envelope Structure Gram + or - Chemical Composition Function
Capsule Both Polysaccharide gel Pathogenicity factor
(Non-essential) Gram + proctecting against
= Slime Gram - phagocytosis until
= Glycocalyx opsonized;
immunogenic
Outer membrane Gram negative Phospholipid/proteins Hydrophobic membrane:
only Lipopolysaccharide LPS=endotoxin
Lipid A Lipid A = toxic moiety
Polysaccharide PS=immunogenic portion

Outer membrane proteins Attachment, virulence, etc.

Proteins porins Passive transport

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 MICROBIOLOGY 1.4
Cell wall=peptidoglycan Gram + (thick) Peptidoglycan open 3-D Rigid support, cell shape, and protection from osmotic damage
Gram – (thin) net of: Synthesis inhibited by penicillins and cephalosporins
N-acetyl-glucosamine Confers Gram reaction
N-acetyl-muramic acid
Amino acids (DAP)

Gram-positive Teichoic acids Immunogenic induces TNF-alpha,IL-1

only Attachment

Acid fast only Mycolic acids Acid-fastness

Resistance to drying and chemicals

Periplasmic space Gram-negative “Storage”space between Enzymes to break down large molecules, (β-lactamases)
only the inner and outer Aids regulation of osmolarity
membranes
Cytoplasmic membrane Gram + Phospholipid bilayer with Hydrophobic cell “sack”
=inner membrane Gram - many embedded proteins Selective permeability and active transport
= cell membrane Carrier for enzymes for:
=plasma membrane Oxidative metabolism
Phosphorylation
Phospholipid synthesis
DNA replication
Peptidoglycan cross linkage
Penicillin Binding Proteins (PBPs)
Pilus or fimbria Primarily Glycoprotein (pilin) Adherence to cell surfaces including attachment to other bacteria during
1. Common Gram – conjugation
2. Sex
3. Virulence
Flagellum + and – Protein (flagellin) Motility

Axial filaments (internal Spirochetes Protein Motilityn

flagellum) Gram -

Table 2. Internal Structures of a Bacterial Cell

Structure Cell type Chemical Composition Function

Nucleoid region Gram + and Gram - DNA Genetic material (all essential genes)
No membrane RNA Primers, mRNA
No histones Proteins Linker proteins, polymerases
No introns

Plasmids Gram + and Gram - DNA Non-essential genetic material

Roles in conjugation, drug resistance, toxin production

Ribosomes Gram + and Gram - 70s (protein/RNA) Protein synthesis

30s (16S RNA)
50s (23 and 5s)

Granules Gram + and Gram - Glycogen, lipids, Storage: polymerization of molecules present in high numbers in cells
polyphosphate, etc. reduces osmotic pressure. Volutin granules of Corynebacterium
diphtheria are used in clinical identification

Endospores Gram + only Keratin coat, calcium Resistance to heat, chemicals, and dehydration
dipicolinate

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 MICROBIOLOGY 1.4

Sample Questions
1. What is the morphology & gram staining reaction of this bacteria? 2. What is the morphology, arrangement & gram reaction of this bacteria

4. What is the morphology of the pink structures?

3. Give the morphology and arrangement of this bacteria: 5. Do you think this is a gram negative bacilli? Why/why not?

6. What is the morphology, gram reaction, and type of flagella? 7. What is this condition and what is its cause?
8. How will you manage this wound with regards to O2 requirement of the bacteria?

9. Which of the following component is present in gram negative bacteria but not in gram positive bacteria?
a. Peptidoglycan b. Flagella c. Lipid A d. Pili e. Capsule

10. Which of the following structures is not part of the bacterial cell envelope?
a. Peptidoglycan b. capsule c. lipopolysaccharide d. Gas vacuole

11. 150 Escherichia coli cells were inoculated into the bladder of a 21 year old female after having sex. These E. coli have a generation time of 20 mins. After a lag of
20 minutes, E. coli enters the logarithmic growth phase . The total # of cells after 2 hours is:
a. 600 b. 1200 c. 2400 d. 4800

12. A 26 year old woman visits her physician because of unusual vaginal discharge. On examination , the physician observes a thin homogenous, white gray discharge
that adheres to the vaginal wall. The pH of the discharge is 5.5 (normal: <4.3) .On gram stain, many epithelial cells covered with gram variable rods are seen
bacterial vaginosis is diagnosed caused by Gardnerella vaginalis. Which one of the following genital normal flora has decreased greatly in numbers, in this
bacterial vaginosis?
a. Corynebacterium b. Staph epidermidis c. Candida albicans d. Lactobacillus spp.

13. Which of the following microorganism is a part of normal vaginal flora but can cause meningitis in newborn?
a. Candida albicans b. Staph. Epidermidis c. Group B Strep ( Strep agalactae) d. Corynebacterium

14. Strep pneumoniae can be part of the normal flora of 5-40% of people. At what anatomic site can it be found?
a. Conjunctiva b. Nasopharynx c. Urethra d. Vagina

Answers: 1. Gram (+) cocci in chain; 2. Cocci in pairs or Diplococci; 3. Gram (+) rod, pleomorphic; 4. Bacilli; 5. No, acid fast staining was used.; 6. Curved rod, gram (-), monotrichous (sinlge
flagellum), 7. Gas gangrene caused by Clostridium perfingens.; 8. Apply Gaseous Atmosphere; 9. C; 10. D; 11. 4800

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