Académique Documents
Professionnel Documents
Culture Documents
microscope and discovered the microbial world. He was a draper (Merchant ) from
Delft, Holland. He used to grind lenses and made microscopes as a hobby. The
microscopes of Leeuwenhoek could magnify objects about 200-300 times. With his
microscopes, Leeuwenhoek observed a variety of things like rain water, pond water
and scrapings from his own teeth. He saw minute moving objects and called them
as Little animalcules, which we now know them as protozoa, yeasts and bacteria.
He made accurate sketches and communicated his findings to Royal Society of
London. Thus, Leeuwenhoek was the first person to discover microscope and the
presence of bacteria and spirochetes in mouth.
THE THEORY OF SPONTANEOUS GENERATION (ABIOGENESIS) After the discovery of
microorganisms by Leeuwenhoek, scientists began investigations about the origin of
microbes. Since organic matter decomposes quickly outside the living body, it was
assumed that microorganisms were arising by spontaneous generation. John
Needham (1749), an Irish priest, observed the appearance of microorganisms in
putrefying meat and interpreted this as spontaneous generation. LA ZARO
SPALLANZANI (1729-1799) - THEORY OF BIOGENESIS Spallanzani, an Italian priest,
boiled beef broth for an hour, sealed the flasks and observed no appearance of
microorganisms and disproved the theory of spontaneous generation or abiotic
origin of Joseph Lister Iwanowsky Metchnikoff Alexander Flemming Paul
Ehrlich Mac Farlane Burnet MEDICAL MICROBIOLOGY life and proposed the theory
of biogenesis. He said that every form of life takes its origin from their parents,
germ cells or seeds. This theory of biogenesis was later proved and supported by
Louis Pasteur. EDWARD JENNER (1749-1823) Jenner was an English country
physician, who discovered a safe and efficient vaccination against small pox. which
ultimately led to the eradication of small pox (Variola). Jenner observed that dairy
workers, exposed to occupational cowpox infection were immune to small pox. He
proved experimentally that resistance to small pox can be induced by injecting cow
pox material (Vaccinia) from disease pustules in to man (in 1796). Pasteur gave the
general term Vaccine (Vacca = cow) in honour of Jenners cow pox vaccine, to
various materials used to induce active immunity. Jenner published his findings in
1798 in a pamphlet An inquiry into the cause and effect of variole vaccine. LOUIS
PASTEUR (1822-1895) He was a Professor of Chemistry at the University of Lille,
France. He is considered as Father of Microbiology, as his contribution led to the
development of Microbiology as a separate scientific discipline. He proved the
theory of Biogenesis and disproved the Theory of spontaneous generation
(Abiogenesis), experimentally by using swan-necked flasks. He worked on souring of
wine and beer and found that this alcohol spoilage is due to the growth of
undesirable organisms, while the desirable microorganisms produce alcohol by a
chemical process called Fermentation. He showed that wine did not spoil, if it is
heated to 50-60C for a few minutes. This method is called Pasteurization, now
widely used in dairy units, to kill pathogenic microorganisms in milk. He is a founder
of Germ theory of disease as he visualized that diseases are caused by
microorganisms. In course of his research, he discovered the importance of
sterilization and discovered steam steri-lizer, autoclave and hot air oven. He also
established the importance of cotton wool plugs for protection of culture media from
aging, since he believed that phagocytes eventually begin to digest the host cells
aided by the effects of intestinal bacteria and that effectively combating them
would increase the life span of human being. HISTORY OF MICROBIOLOGY 7
ALEXANDER FLEMMING (1881-1955) He was an English scientist worked at St.
Marys hospital in London. Flemming was associated with two major discoverieslysozyme and penicillin. In 1922, he doscovered lysozyme by demonstrating that
the nasal secretion has the power of dissolving or lysing certain kinds of bacteria.
Subsequently, he showed that lysozyme was present in many tissues of the body. In
1929, Flemming made an accidental discovery that the fungus Penicillium notatum
produces an antibacterial substance which he called penicillin. Flemming was
culturing Staphylococci in petridishes and some of his cultures were contaminated
with a mold, subsquently indetified as Penicillium notatum. Around the mold colony,
there were clear zones, where Staphylococci disappeared. Flemming attributed this
to the production of an antibacterial susbstance by the mold. Flemming cultured the
fungus Penicilium notatum in broth cultures, filtered the fungal mat and obtained
the penicillin in soluble form in the culture filtrate. In 1940, Howard Florey and Ernst
Chain demonstrated its antibacterial action in vivo. Working in U.S.A., they were
able to produce large quantities of penicillin in pure form. In 1945, Flemming, Florey
and Chain shared the nobel prize in physiology and medicine for the discovery of
penicillin. PAUL EHRLICH (1854-1915) He was a German Bacteriologist, who
pioneered the technique of chemotheraphy in medicine. From his discovery that
certain tissues have a specific affinity, he reasoned that organisms causing diseases
could be selectively killed with chemical drugs. This led him to produce
arsphenamine (an arsenic compound), the first synthetic drug, which destroyed
the syphilis microbe in the body. Ehrlich observed that organic arsenicals killed
trypanosomes in an infected animal, but, if smaller doses were administered, the
trypanosomes acquired tolerance to the drug. Therefore, he aimed at therapia
magna sterilans i.e., the introduction into the blood of a single dose of
chemotherapeutic agent sufficient to kill the parasite. He also observed that drug
would undergo certain changes in the body after it would produce the desired
action. SIR FRANK MAC FARLANE BURNET (1967) Burnet is an Australian scientist,
won nobel prize for the discoery of acquired immunological tolerance. He proposed
clonal selection theory to explain antibody synthesis. His work on bacteriophages
and method for culturing some viruses in live chick embryo, led him to the view that
an animals ability is not inborn, but is developed during fetal life. Burnet (1967)
developed concept of immunological surveillance, according to which the primary
function of the immune system is to preserve the integrity of the body, seeking and
destroying all foreign antigens, whether autogenous or external in origin. Burnets
work also included the mode of action and the epidemiology of influenza virus,
polio, Q fever and the cholera vibrio.
Types of Microbes
Microorganisms are named based on their particular physiological and nutritional characteristics.
The table below provides the name of the microbe with the description.
Physiological
Characteristic Description
Temperature
Psychrophile/
facultative psychrophile
Optimal temperature for growth is 15 C or lower, maximal
temperature is approximately 20 C, and minimal temperature is 0 C
or lower
Psychrotroph
Capable of growing at 5 C or below, with maximal temperature
generally above 25 C to 30 C; term in this case is a misnomer
because it does not indicate nutritional characteristics
Mesophile
Generally defined by optimal temperature for growth, which is
approximately 37 C; frequently grows in the range from 8 C to 10
C and from 45 C to 50 C
Thermophile Grows at 50 C or above
Hyperthermophile
Grows at 90 C or above, although optimal temperature for growth is
generally above 80 C; maximal growth of pure cultures occurs
between 110 C and 113 C, although the maximum (113 C) may
well increase as further research is done
Oxygen
Aerobe
Capable of using oxygen as a terminal electron acceptor; can tolerate
a level of oxygen equivalent to or higher than the 21 percent oxygen
present in an air atmosphere and has a strictly respiratory-type
metabolism
Anaerobe
Grows in the absence of oxygen; some anaerobes have a
fermentative-type metabolism; others may carry out anaerobic
respiration in which a terminal electron acceptor other than oxygen is
used
Facultative anaerobe
Can grow aerobically or anaerobicallycharacteristic of a large
number of genera of bacteria including coliforms such as Escherichia
coli
Microaerophile
Capable of oxygen-dependent growth but only at low oxygen levels;
cannot grow in the presence of a level of oxygen equivalent to that
present in an air atmosphere (21 percent oxygen)
pH
Acidophile Grows at pH values less than 2
Alkalophile Grows at pH values greater than 10
Neutrophile Grows best at pH values near 7
Salinity
Halophile
Requires salt for growth: extreme halophiles (all are archaea), 2.5 M
to 5 M salt; moderate halophiles, usually low levels of NaCl as well
as 15 to 20 percent NaCl
Hydrostatic pressure
(100 atmospheres per
1,000-m depth)
Barophile
Obligate barophiles, no growth at 1 atmosphere of pressure;
barotolerant bacteria, growth at 1 atmosphere but also at higher
pressures. A number of deep-sea bacteria are called barophilic if they
grow optimally under pressure and particularly if they grow
optimally at or near their in situ pressure (0.987 atm = 1 bar = 0.1
megapascal [Mpa])
Nutrition
Autotroph Uses carbon dioxide as its sole source of carbon
Heterotroph Unable to use carbon dioxide as its sole source of carbon and
requires one or more organic compounds
Chemoorganoheterotroph Derives energy from chemical compounds and uses organic
compounds as a source of electrons
Chemolithoautotroph
Relies on chemical compounds for energy and uses inorganic
compounds as a source of electrons. Five classes: hydrogen bacteria,
iron bacteria, sulfur bacteria, ammonia oxidizers, and nitrite
oxidizers. Specific nutritional groups of bacteria that do not clearly
fit in this category include obligate methane oxidizers and the carbon
monoxide oxidizers. There are also photoorganoheterotrophs and
photolithoautotrophs among the anoxigenic photosynthetic bacteria.
Mixotroph
Capable of growing both chemoorganoheterotrophically and
chemolithoautotrophically; examples include some of the hydrogen
bacteria and some species of Thiobacillus (sulfur-oxidizing bacteria)
Oligotroph
Can develop at first cultivation on media containing minimal organic
material (1 to 15 micrograms carbon per liter) and grow on such
media in subsequent cultivation
Copiotroph Requires nutrients at levels 100 times those of oligotrophs
Microbes are far more likely than multicellular organisms to retain viability on small solar
system bodies because they can adapt to a much wider range of environmental conditions.
Single-cell organisms have infiltrated virtually every corner of Earth's biosphere and still
constitute the bulk of the earth's biomass. They grow in temperate marine and terrestrial settings,
within other microbial or multicellular organisms, in deep subsurface niches, and in extreme
environments that would be lethal for other life forms. They often influence geochemical
reactions within the biosphere and frequently play key roles in food webs and complex
ecosystems.
The physiological state of microorganisms influences their ability to survive extreme
environmental conditions. For example, organisms with active DNA repair mechanisms or a
protective layer of material are more likely to endure exposure to ultraviolet (UV) or ionizing
radiation than are cells without equivalent capabilities. Viable microorganisms are either
cell-wall polymers, including teichoic acids, teichuronic acids and proteins, are also
present. Gram-negative bacteria have a thinner peptidoglycan layer and an
additional outermembrane that differs in structure from the cytoplasmicmembrane
(Figure 1.4b). The outer membrane contains lipopolysaccharides on its outer face,
phospholipids on its inner face, proteins and lipoproteins which anchor it to the
peptidoglycan. Porins are a group of proteins that form channels through which
small hydrophilic molecules, including nutrients, can cross the outer membrane.
Lipopolysaccharides are Medical Microbiology and Infection Lecture Notes, Fifth
Edition. Edited by Tom Elliott, Anna Casey, Peter Lambert and Jonathan Sandoe.
2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd. a
characteristic feature of Gram-negative bacteria and are also termed endotoxins or
pyrogen. Endotoxins are released on cell lysis and have important biological
activities involved in the pathogenesis of Gram-negative infections; they activate
macrophages, clotting factors and complement, leading to disseminated
intravascular coagulation and septic shock (Chapter 33). Red blood cell Bacillus
anthracis Clostridium perfringens Escherichia coli Borrelia recurrentis Treponema
pallidum Staphylococcus aureus Streptococcus pneumoniae Chlamydia trachomatis
Mycoplasma pneumoniae 0 5 Size (m) 10 Figure 1.1 Shape and size of some
clinically important bacteria. 4 Basic bacteriology Mycobacteria have a distinctive
cell wall structure and composition that differs from that of Gram-positive and
Gram-negative bacteria. It contains peptidoglycan but has large amounts of high
molecular weight lipids in the form of long chain length fatty acids (mycolic acids)
attached to polysaccharides and proteins. This high lipid content gives the
mycobacteria their acid fast properties (retaining a stain on heating in acid), which
allows them to be distinguished from other bacteria (e.g. positive Ziehl-Neelsen
stain). The cell wall is important in protecting bacteria against external osmotic
pressure. Bacteria with damaged cell walls, e.g. after exposure to b-lactam
antibiotics such as penicillin, often rupture. However, in an osmotically balanced
medium, bacteria deficient in cell walls may survive in a spherical form called
protoplasts. Under certain conditions some protoplasts can multiply and are referred
to as L-forms. Some bacteria, e.g. mycoplasmas, have no cell wall at any stage in
their life cycle. The cell wall is involved in bacterial division. After the nuclear
material has replicated and separated, a cell wall (septum) forms at the equator of
the parent cell. The septum grows in, produces a cross-wall and eventually the
daughter cells may separate. In many species the cells can remain attached,
forming groups, e.g. staphylococci form clusters and streptococci form long chains
(Figure 1.5). Capsules Some bacteria have capsules external to their cell walls
(Figure 1.3). These structures are bound Coccus Curve Spiral Bacillus (rod) Figure
1.2 Some bacterial shapes. DNA Cytoplasm Capsule Cell wall Flagellum Fimbriae
Inclusion granules Ribosomes Cross-wall forming Cytoplasmic membrane Figure 1.3
A section of a typical bacterial cell. Basic bacteriology 5 to the bacterial cell and
have a clearly defined boundary. They are usually polysaccharides with
characteristic compositions that can be used to distinguish between microorganisms
of the same species (e.g. in serotyping). Capsular antigens can be used to
differentiate between strains of the same bacterial species, e.g. in the typing of
Streptococcus pneumoniae for epidemiological purposes. The capsules are
important virulence determinants in both Gram-positive and Gram-negative
bacteria, because they may protect the bacteria from host defences and, in some
bacteria, aid attachment to host cells. Bacterial slime and biofilm Extracellular slime
layers are produced by some bacteria. They are more loosely bound to the cell
surface than capsules and do not form a clearly defined surface boundary. The slime
layer is composed predominantly of complex polysaccharides (glycocalyx), which
acts as a virulence Figure 1.4 Cell wall and cytoplasmic membrane of (a) Grampositive bacteria, (b) Gram-negative bacteria and (c) mycobacteria. The Grampositive bacterial cell wall has a thick peptidoglycan layer with associated molecules
(teichoic acids, teichuronic acids and proteins). The Gram-negative bacterial cell
wall contains lipopolysaccharides, phospholipids and proteins in an outer membrane
linked to a thin inner peptidoglycan layer. The mycobacterial cell wall contains long
chain length fatty acids (mycolic acids). Staphylococci Neisseriae Pneumococci
Streptococci Figure 1.5 Some groups of bacteria. 6 Basic bacteriology factor through
the formation of biofilm, e.g. by facilitating the attachment of Staphylococcus
epidermidis onto artificial surfaces, such as intravascular cannulae (Figure 1.6),
replacement joints and heart valves. Once formed, biofilms present a major problem
for treatment and may require removal of the biomedical device. Flagella Bacterial
flagella are spiral-shaped surface filaments consisting mainly of the protein,
flagellin. They are attached to the cell envelope as single (monotrichous) or multiple
(peritrichous) forms (Figure 1.7). Flagella facilitate movement (motility) in bacteria
by rapid rotation. They can be observed under the light microscope with special
stains. Flagella are usually detected for diagnostic purposes by observing motility in
a bacterial suspension or by spreading growth on solid media. The antigenic nature
of the flagella may be used to differentiate between and identify strains of
Salmonella spp. Fimbriae Fimbriae (also termed pili) are thin, hair-like appendages
on the surface of many Gram-negative, and some Gram-positive, bacteria (Figure
1.3). They are approximately half the width of flagella, and are composed of
proteins called pilins. In some bacteria they are distributed over the entire cell
surface. Fimbriae are virulence factors enabling bacteria to adhere to particular
mammalian cell surfaces, an important initial step in colonisation of mucosal
surfaces, e.g. Neisseria gonorrhoeae produce fimbriae that bind to specific receptors
of cervical epithelial cells, whereas Streptococcus pyogenes have fimbriae
containing M protein, which facilitates adhesion to human cells in the pharynx.
Specialised fimbriae are involved in genetic material transfer between bacteria, a
process called conjugation. Figure 1.6 Scanning electronmicrograph of
Staphylococcus epidermidis embedded in slime attached to a catheter.
Monotrichous Peritrichous Figure 1.7 Arrangements of bacterial flagella. Basic
bacteriology 7 Intracellular structures Nuclear material The bacterial chromosome
consists of a single circular molecule of double-stranded DNA, which is maintained
in a compact form within the cell by supercoiling. When released from the cell and
uncoiled the DNA would be about 1 mm long (10 to 100-times the length of the
cell). Additional smaller extra-chromosomal DNA molecules, called plasmids, may
also be present in bacteria. The chromosome usually codes for all the essential
functions required by the cell; some plasmids control important phenotypic
properties of pathogenic bacteria, including antibiotic resistance and toxin
production. Extracellular nuclear material for encoding virulence and antibiotic
resistance may also be transferred between bacteria and incorporated into the
microbiology and in understanding the tests for identifying bacteria. Carbon and
nitrogen sources Bacteria are classified into two main groups according to the type
of compounds that they can utilise as a carbon source: 1 Autotrophs utilise
inorganic carbon from carbon dioxide and nitrogen from ammonia, nitrites and
nitrates; they are of minor medical importance. 2 Heterotrophs require organic
compounds as their major source of carbon and energy; they include most bacteria
of medical importance. Atmospheric conditions Carbon dioxide Bacteria require CO2
for growth; adequate amounts are present in the air or are produced during
metabolism by the microorganisms themselves. A few bacteria, however, require
additional CO2 for growth, e.g. Neisseria meningitidis, Campylobacter jejuni.
Oxygen Bacteria may be classified into four groups according to their O2
requirements: 1 Obligate (strict) aerobes: grow only in the presence of oxygen, e.g.
Pseudomonas aeruginosa. 2 Microaerophilic bacteria: grow best in low oxygen
concentrations, e.g. Campylobacter jejuni. 3 Obligate (strict) anaerobes: grow only
in the absence of free oxygen, e.g. Clostridium tetani. 4 Facultative anaerobes: grow
in the presence or absence of oxygen, e.g. Escherichia coli. Temperature Most
pathogenic bacteria grow best at 37 C. However, the optimum temperature for
growth is occasionally higher, e.g. for C. jejuni, it is 42 C. The ability of some
bacteria to grow at low temperatures (04 C) is important in food microbiology;
Listeria monocytogenes, a cause of food poisoning, will grow slowly at 4 C and has
resulted in outbreaks of food poisoning associated with cookchill products. pH Most
pathogenic bacteria grow best at a slightly alkaline pH (pH 7.27.6). There are a few
exceptions: Lactobacillus acidophilus, present in the Figure 1.9 Gram-stain of
Clostridium sporogenes (showing oval subterminal spores) and a Clostridium tetani
with a terminal spore (arrowed). Basic bacteriology 9 vagina of post-pubescent
females, prefers an acid medium (pH 4.0). It produces lactic acid, which keeps the
vaginal secretions acid, thus preventing many pathogenic bacteria from establishing
infection. Vibrio cholerae, the cause of cholera, prefers an alkaline environment (pH
8.5). Growth in liquid media When bacteria are added (inoculated) into a liquid
growth medium, subsequent multiplication can be followed by determining the total
number of live microorganisms (viable counts) at various time intervals. The growth
curve produced normally has four distinct phases (Figure 1.10): 1 Lag phase (A): the
interval between inoculation of a fresh growth medium with bacteria and the
commencement of growth; 2 Log phase (B): the phase of exponential growth; the
growth medium becomes visibly turbid at approximately 1 106 cells/ml; 3 Stationary
phase (C): the growth rate slows as nutrients become exhausted, waste products
accumulate, and the rate of cell division equals the rate of death; the total viable
count remains relatively constant; 4 Decline phase (D): the rate of bacterial division
is slower than the rate of death, resulting in a decline in the total viable count. Note
that the production of waste products by bacteria, particularly CO2, and the uptake
of O2 have been utilised in the development of semiautomated instruments to
detect bacterial growth in blood samples obtained from patients with suspected
bloodstream infection. Growth on solid media Liquid growth media containing the
nutrients needed for bacterial growth can be solidified with agar, a polysaccharide
extracted from seaweed. Heating during sterilisation of the medium melts the agar,
which then remains liquid until the temperature falls to approximately 40 C, when it
produces a transparent solid gel. Solid media are normally set in Petri dishes (agar
plates). When spread across the surface of an agar plate, most bacteria grow as
visible colonies. Each colony comprises millions of bacterial cells that emanated
from either a single cell or a cluster of cells. The appearance of the bacterial colony
(colonial morphology) assists in identification. Growth on laboratory media To grow
bacteria in vitro, the microbiologist has to take into account the physiological
requirements. Various types of liquid and solid media have been 0 12 Time (h)
Viable count (per mL) Bacteria inoculated A 102 104 106 108 1010 B C D 24 Figure
1.10 Bacterial growth curve showing the four phases: (A) lag; (B) log or exponential;
(C) stationary; and (D) decline (death). 10 Basic bacteriology developed for the
diagnostic microbiology laboratory. Simple media Many bacteria will grow in or on
simple media, e.g. nutrient broth/nutrient agar that contains peptone
(polypeptides and amino acids from the enzymatic digestion of meat) and meat
extract (water-soluble components of meat containing mineral salts and vitamins).
Enriched media These contain additional nutrients for the isolation of more
fastidious bacteria that require special conditions for growth, e.g. agar containing
whole blood (blood agar) or agar containing lysed blood (chocolate agar). Selective
media These are designed to facilitate growth of some bacteria, while suppressing
the growth of others, and include: . mannitol salt agar which contains increased
NaCl (salt) concentration for the recovery of staphylococci; . MacConkey agar, which
contains bile salts and allows the growth of bile-tolerant bacteria only; and .
antibiotics, which are frequently added to media to allow only certain bacteria to
grow while suppressing or killing others. Indicator media These are designed to aid
the detection and recognition of particular pathogens. They are often based on
sugar fermentation reactions that result in production of acid and the subsequent
colour change of a pH indicator, e.g. MacConkey agar contains lactose and a pH
indicator (neutral red); lactose-fermenting bacteria (e.g. Escherichia coli) produce
acid and form pink colonies, whereas non-lactose fermenting bacteria (e.g.
Salmonella spp.) do not produce acid and form pale yellow colonies. This property
facilitates the recognition of possible Salmonella colonies among normal bowel flora.
Note that indicator media may also contain selective agents including antibiotics or
substances such as bile salts and crystal violet to suppress growth of most Grampositive microorganisms. MacConkey agar is therefore both a selective medium and
an indicator medium. Basic bacteriology 11