Class no 2.

MICROFLORA OF HUMANS
Physiologically free of microorganisms:
- fetus (germ-free in utero; microbes begin colonization of the body soon after birth),
- bodily fluids (blood, lymph, CSF),
- most internal tissues and organs (CNS, lower bronchi and alveoli, liver, spleen, kidneys, bladder).
IMPORTANT CONCEPTS
Carrier state – an individual harbors the potential pathogen („true pathogen”) and therefore serves as a source
of infection for others (in asymptomatic infections and in recovery state).
Colonization – the acquisition of a new organism, which then may cause a disease or be eliminated by the host
defenses.
Opportunistic pathogens – starts out the commensalist, then changes to the parasite , e.g. Escherichia coli
WHY ARE WE COLONIZED BY MICROBES?
- Excellent conditions for proper growth (moisture, pH, temperature, nutrients).
- Conditions vary throughout the body (different flora in different places).
- Diverse composition of the human microbiome (genetics, age, sex, stress, antimicrobial agents, disruption
of normal anatomic or physiologic function, nutrition and diet of the individual).
RESIDENT FLORA VS TRANSIENT FLORA
Resident flora – relatively fixed group of microorganisms regularly found in a given area at a given age.
If disturbed, it promptly reestablish itself.
Transient flora – present temporally, but generally not disease-causing. When normal flora is disturbed,
the transient flora may cause disease.
ADVANTAGES OF THE PRESENCE OF NORMAL MICROFLORA
- Colonization resistance (prevent colonization by pathogens).
- Nutritional function (vitamin B and K producers, e.g. E. coli).
- Secretion of noxious substances (short-chain fatty acids, bacteriocins, lactic acid).
- Stimulation of the immune system.
- May antagonize other bacteria by: limiting nutrients (competitive exclusion), limiting space, altering
the environment in ways which inhibit the growth of other microbes (microbial antagonism).
DISADVANTAGES OF THE PRESENCE OF NORMAL MICROFLORA
- Harmful for the immunocompromised and debilitated individuals.
- Harmful if present in abnormal anatomic locations (due to surgery or accident).
NORMAL FLORA OF SKIN
30 different microbial types, primarily bacteria 10,000-100,000 bacterial cells/cm² (Gram-positive anaerobes)
and fungi can be found on the surface of the skin (stratum corneum) and deeper layers (hair follicles, sweat,
and sebaceous glands), including: Staphylococcus, Micrococcus, Streptococcus, Corynebacterium,
Propionibacterium, Brevibacterium, Acinetobacter, Lactobacillus, Candida, Pityrosporum.
NORMAL FLORA OF EARS AND EYES
Middle ear and inner ear usually are sterile. Outer ear and the auditory canal (moist areas) may have similar
microflora as in the mouth or nose (staphylococci, streptococci, diphtheroids). External surface of the eye
s lubricated, cleansed, and protected by tears, mucus, and sebum (lysozyme activity) Eye microflora:
Staphylococcus, Streptococcus, Corynebacterium, Moraxella catarrhalis.
NORMAL FLORA OF ORAL CAVITY
Gum margins, crevices between the teeth, tonsil crypts contain microorganisms; particles of food, dead
epithelial cells around the teeth help microbes to multiply. As a result of bacterial activity dental caries (tooth
decay), gingivitis (gum disease), periodontal diseases may develop. Gram-positive and Gram-negative cocci,
Gram-positive and Gram-negative bacilli, spirochetes (e.g. Actinomyces, Bacteroides, Fusobacterium,

1
Lactobacillus, Porphyromonas, alpha-hemolytic streptococci, Neisseria, Veillonella, Treponema,
Peptostreptococcus) are found in the oral cavity.
NORMAL FLORA OF GASTROINTESTINAL TRACT
Esophagus – quick food passage stomach – extremely acidic pH, gastric enzymes (but Helicobacter pylori
and Mycobacterium tuberculosis can survive).
Small intestine: the duodenum – slight microflora (bile presence and peristalsis); the jejunum and ileum
– staphylococci, streptococci, lactobacilli, Veillonella, Clostridium, yeasts.
Large intenstine: colon – 10 ¹¹ bacterial cells/g feces, 500-600 species (Bacteroides, Fusobacterium,
Bifidobacterium, Eubacterium, Clostridium, Peptostreptococcus, Veillonella, Enterobacteriaceae, Enterococcus,
Streptococcus, Pseudomonas, Lactobacillus, Mycoplasma, Candida).
NORMAL FLORA OF RESPIRATORY TRACT
Upper respiratory tract (nasal passages and the throat/pharynx): streptococci, staphylococci, diphtheroids,
lactobacilli, micrococci, Neisseria, Haemophilus.
Lower respiratory tract is germ-free (larynx, trachea, bronchi, bronchioles, and lungs).
Defense mechanisms in respiratory tract are: ciliated epithelium, coughing, sneezing, swallowing.
NORMAL FLORA OF URINARY TRACT
Healthy kidneys, ureters, and urinary bladder (acidic urine) are sterile; urine flow, pH are protective mechanisms.
In distal urethra and external opening of the urethra: Neisseria, staphylococci, streptococci, enterococci,
diphtheroids, mycobacteria, mycoplasmas, enteric/intestinal Gram-negative rods, anaerobes, yeasts can
be found.

NORMAL FLORA OF GENITAL TRACT

The reproductive system ( ) is usually sterile, with the exception of the vagina.
Vaginal microflora varies with the stage of sexual development:
- puberty and following menopause (alkaline secretions) - diphtheroids, streptococci, staphylococci,
and coliforms (enteric rods).
- childhood (acidic secretions), adult women - lactobacilli, alpha-hemolytic streptococci, staphylococci,
diphtheroids, yeasts.
ENVIRONMENTAL FACTORS AFFECTING BACTERIA
1. TEMPERATURE
- In high temperature (>90°C) – denaturation of bacterial proteins, destruction of nucleic acids, and cellular
membranes takes place.
- In low temperature (0-7°C) – reduction of microbial metabolic rate (reproduction or toxin synthesis
are impossible) is observed; this is microstatic effect (psychrotrophs are exceptions).
- Freezing – slow freezing (-20°C): ice crystals disrupt cellular and molecular structure of bacteria and fungi;
quick freezing (-70°C): microbes are dormant/not killed.
2. RADIATION
- Ionizing radiation (gamma, X, high-energy electron beams) – there is water ionization; highly reactive
hydroxyl radicals are formed, reacting with organic cellular components (esp. DNA).
- Non-ionizing radiation (UV, 230-270 nm) – not penetrating; damage of bacterial DNA by causing bonds
between adjacent pyrimidine bases (thymine dimers); DNA replication is inhibited. Sunlight contains some
UV radiation but it is screened by the ozone layer; formation of very reactive singlet oxygen (O2) in bacterial
cytoplasm. The effect depends on wavelength, intensity, and duration.
3. DESSICATION
- Drying out (absence of water) /desiccation – bacterial growth and reproduction are slowed down.
- High/low pressure – proteins and carbohydrates are altered (rapid inactivation of vegetative cells). Osmotic
pressure (salts/sugars), ultrasounds, pH.
PREVENTION OF INFECTION
2
- Sanitation – process by which microbial contamination is brought to a "safe" level. This process refers
primarily to the process of "cleaning" inanimate objects.
- Asepsis – prevention of contact with microorganisms.
- Antisepsis – process that leads to the removal of microorganisms from skin, mucous membranes and wounds
using antiseptics .
- Antiseptics – antimicrobial substances that are applied to living tissue/skin to reduce the possibility
of infection or putrefaction.
- Disinfection – process that reduces or eliminates all pathogenic microorganisms except spores.
- Disinfectants – substances killing vegetative forms of microorganisms; do not eliminate bacterial and fungal
spores, and non-lipid viruses; used for disinfection of articles - medical instruments, bed-clothes, air, hands,
excrements.
- Sterilization – the process by which an article, surface or medium is made free of all microorganisms,
including spores, viruses, and fungi, that are removed or killed/inactivated. Methods of sterilization: Physical,
high temperature, ionizing radiation, filtration. Chemical sterilization possible but the chemicals must be used
in high concentration for a long time (formaldehydes, glutaraldehydes, dyes, halogens, phenols, ethylene
oxide).
- Sterile - term is absolute and indicates complete freedom from all microorganisms and their spores.
STERILIZATION METHODS
1. Sterilization with high temperature
A. “Dry heat” procedures – kill microorganisms due to organic compounds oxidation; for heat-tolerant articles
(e.g. flaming to red heat, hot air oven, incineration):
- Sterilization in an open flame (temperature not controlled) e.g, burning, flaming, annealing of wire-loop.
- Sterilization in a heater (temperature controlled: 180°C, 2-3 hrs) - sterilization of the equipment resistant
to high temperatures e.g., Petri dishes, tubes, pipettes, small instruments.
B. “Moist heat” procedures (autoclaving) – more efficient than „dry heat” procedures, presence of hot water
causes protein denaturation resulting in disruption of cell membranes, and improves heat penetration
(e. g. autoclaving, tyndallization); Steam under pressure is the main killing factor for all microorganisms
(protein denaturation), including vegetative bacterial forms, bacterial and fungal spores, and viruses. To sterilize
surgical instruments 134-138°C is used; for bacteriological media dressings and rubber gloves 121°C is used;
The time of sterilization depends on the type of material and amount of load in the autoclave. Autoclaving
has to be controlled. Each sterilization cycle must be monitored by continuous recording of the temperature,
time, and steam pressure. Items loaded into the autoclave should be covered or closed to prevent
recontamination after autoclaving. Efficacy of autoclaving has to be tested using chemical methods (browne’s
tube - green spot) or biological methods (e.g. Bacillus stearothermophilus - Sporal A and Bacillus subtilis - Sporal
S).
2. TYNDALLIZATION
In the Koch apparatus (three times pasteurization); the material is heated to the temperature of boiling water
(100°C) for 60 min, three times, day after day. It is used to sterilize bacteriological media that cannot be treated
with the temperature above 100°C. Result: germ-free material.
3. RADIATION„COLD STERILIZATION”
X-rays and gamma-rays (for 48hrs) penetrate through various materials and kill vegetative bacterial and fungal
cells, and bacterial and fungal spores, despite their external protection. Used for plastic, glass, catheters, oils,
fabrics.
4. STERILIZATION WITH UV LIGHT
UV-C light - germicidal UV - kill or inactivate microorganisms by destroying nucleic acids/DNA at wavelengths
~250 nm - 260 nm; UV breaks molecular bonds within DNA, producing thymine dimers. "Sterilization"
theoretically possible/controlled environment misquoted as being achievable: where there are obstacles that
block the UV light; a phenomenon known as light and dark repair photoreactivation and base excision repair.
3
5. STERILIZATION BY FILTRATION
Filters are made of various porous materials, nowadays usually from esters of cellulose (membrane filters) pore
size: 0.2 µm or below; It is used to remove microorganisms from heat labile liquids, fluids (sera, sugars,
antibiotics for culture media).
6. STERILIZATION BY ETHYLENE OXIDE
Ethylene oxide (EtO) is used to sterilize medical and pharmaceutical products that cannot support conventional
high temperature steam sterilization - devices that incorporate electronic components, plastic packaging, plastic
containers. EtO gas infiltrates packages as well as products themselves to kill microorganisms. Mixed with air
at a ratio of at least 3% EtO gas, forms an explosive mixture. For safety - mixed with nitrogen or CO2; there
is intrinsic safe material (ATEX) zoning used, for security of people as well as security of the process itself.
The overall duration of this cycle is around 60 hours.
DISINFECTION METHOD
Methods of disinfection: chemical and physical (temperature, ultrasounds).
1. PASTEURISATION (DISINFECTION WITH HIGH TEMPERATURE)
Performed by the heating of material (usually food) to a temp. of 65°C for 30 min, then quickly cooling
to a temp. of 4°C. Most vegetative bacterial forms are killed but thermophiles and spores survive (method
not used in medicine!). Used for milk, ice-cream, yogurt, and fruit juices, beer, wine.
UHT STERILIZATION of food by heating it for an extremely short period, around 1-4 seconds, at a temp. 135°C
to 150°C, and then cooling it rapidly. UHT sterilization principle: the irreversible change of bacterial protein, then
some globulins can't be dissolved and the enzymes lose power, thus cause the loss of metabolism ability.
Advantages of UHT sterilization: Long shelf life, cheaper packaging. Disadvantages: changed structures
of proteins in milk.
2. DECOCTION (DISINFECTION WITH HIGH TEMPERATURE)
Boiling (100°C, 10 min); immediately kills vegetative bacterial and fungal forms and viruses, except for hepatitis
viruses. Addition of 2% sodium carbonate – sporicidal effect.
3. CHEMICALS USED IN DISINFECTION
Organic alcohols, aldehydes, phenols, carbolic acid, resorcinol, thymol, acids and bases, salts, oxidizing agents,
dyes, soap, detergents, gaseous formaldehyde, SO2, ozone, chlorine, fluorine.
Chemicals used as disinfectants:
- High level disinfectants (glutaraldehyde, hydrogen peroxide, chlorine compounds) may be used
for endoscopes, cystoscopes, surgical instruments with plastic components,
- Intermediate level disinfectants (alcohols, phenols): may be used for laryngoscopes, fiber optic endoscopes,
- Low level disinfectants (soap, detergents): may be used for stethoscopes, ECG electrodes.
MECHANISMS OF DISINFECTANTS AND ANTISEPTICS ACTION
- Protein denaturation - alcohols,
- Cell wall and outer cellular membrane disruption - glutaraldehyde, EDTA and other permeabilizers
- Inner cytoplasmic membrane - QACs (quaternary ammonium compounds), chlorhexidine, diamines,
alexidine, phenols,
- Cross-linking of macromolecules - formaldehyde, glutaraldehyde,
- DNA intercalation - acridines,
- Interaction with thiol groups - silver compounds,
- Effects on DNA - halogens, hydrogen peroxide, silver ions,
- Oxidizing agents - halogens, peroxygens (hydrogen peroxide).
FACTORS THAT INFLUENCE THE EFFICACY OF DISINFECTION/STERILIZATION
- Concentration – in general, the more concentrated the disinfectant, the greater its efficacy and the shorter
the time necessary to achieve microbial kill;
- Contact time

4
- pH – an increase in pH improves the antimicrobial activity of some disinfectants (e.g. glutaraldehyde,
quaternary ammonium compounds) but decreases the antimicrobial activity of others (e.g. phenols,
hypochlorites, and iodine);
- Temperature – the activity of most disinfectants increases as the temperature increases;
- Nature of microorganism – spores are resistant to disinfectants (spore coat and cortex), Mycobacteria have
a waxy cell wall, Gram-negative bacteria possess an outer membrane;
- Number of microorganism (bioburden) – 30 minutes to kill 10 B. atrophaeus (formerly B. subtilis) spores but
3 hours to kill 100,000 B. atrophaeus spores. The need for scrupulous cleaning!
- Presence of organic material – serum, blood, pus, or fecal or lubricant material; interference occurs
by a chemical reaction between the germicide and the organic matter or as physical barrier;
- Material composition
RELATIVE MICROBIAL RESISTANCE TO PHYSICAL AND CHEMICAL FACTORS
The most resistant forms are prions, then bacterial spores (e.g. endospores of Clostridium difficile),
Mycobacterium sp., non-lipid and small viruses (e.g. Poliovirus), fungi (e.g. Candida spp.), lipid and big viruses
(e.g. HIV), vegetative forms of bacteria (Staphylococcus spp.).
COMPARISON OF PROKARYOTIC AND EUKARYOTIC CELL ORGANIZATION
EUKARYOTES PROKARYOTES
• nucleus: present absent
• number of chromosomes: more than one one (not true) + plasmids
• cell type: usually multicellular usually unicellular
• nuclear membrane: present absent
• example: animals and plants bacteria and archaea
• genetic recombination: meiosis/fusion of gametes binary fission („division in half”)
• lysosomes present absent
• microtubules: present absent or rare
• endoplasmic reticulum: present absent
• mitochondria: present absent
• cytoskeleton: present may be absent
• histones: present absent
• ribosomes: larger (80S) smaller (70S)
• Golgi apparatus: present absent
• chloroplasts: present (in plants) absent
• cell wall: in plant cells and fungi complex
• cell size: 10-100µm 1-10µm
PROKARYOTIC CELL STRUCTURE
CYTOPLASMIC STRUCTURES
Have no organelles, no membrane-enclosed nucleus (nucleoid), and no histones; in rare cases, they contain
complex phospholipids, sphingolipids, and sterols (Mycoplasma). Are haploid with a single chromosome. Have
70S ribosomes composed of 30S and 50S subunits. Have a cell wall composed of peptidoglycan-containing
muramic acid. Certain genera, such as Bacillus and Clostridium, produce endospores in response to harsh
environmental conditions. Endospores are small, dormant (inactive), asexual spores that develop inside bacterial
cell (active vegetative cell). Endospores should not be confused with reproductive spores of fungi. Endospores
are visualized microscopically as unstained areas in cell with the use of Gram stain or by using specifics pore
stains (Schaeffer-Fulton).
PLASMA MEMBRANE
The plasma membrane (PM) is phospholipid bilayer with embedded proteins. The prokaryotic PM is made
of phospholipids and proteins but does not contain sterols, in contrast to eukaryotic PMs (except
5
for Mycoplasma). The PM acts as an osmotic barrier and is the location of the electron transport chain, where
energy is created.
CELL WALL
Cell wall is a rigid structure that maintains the shape of the cell. Types of cell walls are categorized according
to their staining characteristics: Gram-positive and Gram-negative. Although they stain Gram-positive,
mycobacteria have a modified cell wall called acid fast. Mycoplasmas are microorganisms that have no cell wall.
Gram-Positive Cell Wall is composed of a very thick protective peptidoglycan (murein) layer. The peptidoglycan
layer consist of glycan chains of alternating N-acetyl-d-glucosmine (NAG) and N-acetyl-D-muramic acid (NAM).
Teichoic acid (anchored to the murein) and lipoteichoic acid (anchored to the PM) are unique to the Gram-
positive cell wall.
Gram-Negative Cell Wall is composed of two layers. The inner peptidoglycan layer is much thinner in Gram-
postitive cell walls. Outside the peptidoglycan layer is additional outer membrane unique to the Gram-negative
cell wall. The outer membrane contains proteins, phospholipids and lipopolysaccharide (LPS). LPS contains three
regions: an antigenic O-specific polysaccharide, a core polysaccharide, an inner lipid A (also called endotoxin).
The lipid A is responsible for producing fever and shock conditions in patients infected with Gram-negative
bacteria. Between the outer membrane and the inner membrane is the periplasmic space.
Acid-Fast Cell Wall. Certain genera, such as Mycobacterium and Nocardia, have a Gram-positive cell wall
structure but also contain a waxy layer of glycolipids and fatty acids (mycolic acid). Mycobacteria and nocardiae
are best stained with an acid-fast stain (Ziehl-Neelsen).
Absence of cell wall. Mycoplasma and Ureaplasma lack a cell wall and contain sterols in their cell membranes.
SURFACE POLYMERS
Capsule is made of polysaccharide polymers or of polypeptides. Capsules act as virulence factors in helping
the pathogen evade phagocytosis.
Slime layer is similar to capsule but is more diffuse layer surrounding the cell.
CELL APPENDAGES
Flagella (plural of flagellum) are the organs of locomotion. Flagella are exterior protein filaments that rotate
and cause bacteria to be motile.
Pili (plural of pilus) are nonmotile, long, hollow protein tubes that connect two bacterial cells and mediate DNA
exchange.
Fimbriae (plural of fimbria) are proteinaceous, hairlike appendages that adhere some bacterial cells to one
another and to environmental surfaces.
MICROSCOPIC SHAPES
BACTERIAL SHAPE
Bacteria vary in size from 0,4 to 2 µm. They occur in three basic shapes:
• cocci (spherical), e.g. Staphylococcus sp.
• bacilli (rod-shaped), e.g. Escherichia coli
• spirochetes (spiral), e.g. Treponema pallidum
When a species varies in size and shape within a pure culture, the bacterium is pleomorphic.
BACTERIAL ARRANGEMENT
• Chains (cocci, rods), eg. Streptococcus pyogenes
• Pairs (cocci, rods), e.g. Neisseria meningitidis
• Cubical bundles (cocci), e.g. Staphylococcus sp.
• Flat plates
MICROBIAL NUTRITION
All bacteria have three major nutritional needs for growth:
• A source of carbon (for making cellular constituents). Carbon represents 50% of the dry weight of bacterium.
• A source of nitrogen for making proteins. Nitrogen makes up 14% of the dry weight.

6
• A source of energy (adenosine triphosphate [ATP], for performing cellular functions.
IDENTIFICATION OF BACTERIA
Identification of bacteria is based on:
1. Cellular morphology after staining.
2. Motility resulting from the presence of flagella.
3. Presence or absence of endospores and other cellular elements.
4. Growth characteristics:
- rapidity,
- morphology in solid and liquid culture media,
- optimal atmospheric conditions,
- temperature of incubation,
- colonial features.
5. Results of differential biochemical reactions.
6. Results of serological tests.
7. Results of nucleic acid homology studies.

DIRECT/INDIRECT VISUALISATION OF MICROORGANISMS

OPTICAL METHODS
1. Bright-Field Microscopy (Light Microscopes) – useful magnification (makes visible the smallest resolvable
particles) = power of objective x power of ocular (microorganisms are observed under 1000 magnification);
2. Dark-Field Microscopy – the eye reaches only the light reflected - from the object examined (spirochetes);
3. Phase Contrast Microscopy – light waves passing through bacteria emerge in different phases; optical system
converts difference in phases into difference in intensity (living cells);
4. Fluorescent Microscopy (e.g. fluorescein-labeled antibodies);
5. Electron Microscopy – most precise view of microbes, esp. their inner and outer structures
(for all microorganisms, including viruses);
STAINING METHODS
1. Negative Staining – staining of the background with an acid dye (nigrosin); cells/capsules – in contrast – are
colourless;
2. Positive Staining – Simple: one stain; Complex: two or more stains (e.g. Gram stain, acid-fast stain);
3. Positive-Negative Staining – Staining of the background with an acid dye and cells with contrasting stain;
capsules – in contrast – are colourless.
Gram staining is an example of complex positive staining.
GRAM STAINING PROCEDURE
• Stain with crystal violet for 1 minute,
• Gently wash of the stain with tap water,
• Gently apply Gram’s iodine for 1 minute,
• Gently wash of the stain with tap water,
• Add the alcohol (decolourizer) for 30 seconds,
• Counterstain with safranin (or fuchsin) for 1 minute,
• Gently wash of the stain with tap water,
• Dry with bibulous paper.
Acid-fast staining is an example of complex positive staining.
1. Ziehl-Neelsen method for acid-fast staining – for bacteria that retain carbol fuchsin after decolourization
with hydrochloric acid in alcohol; e.g. Mycobacterium sp., Actinomyces sp. Also for bacterial endospores!
ZIEHL-NEELSEN STAINING PROCEDURE
7
• Flood slide with carbol fuchsin,
• Hold a flame beneath the slide until steam appears but do not allow it to boil,
• Allow hot slide to sit for 3 to 5 minutes, rinse with tap water,
• Flood slide with 3% hydrochloric acid in isopropyl alcohol,
• Allow to sit 1 minute, rinse with tap water,
• Flood slide with methylene blue,
• Allow to sit 1 minute, rinse with tap water,
• Blot dry and view under oil immersion lens.
2. Kinyoun method for acid-fast staining;