Microbiology

The study of microorganisms

The discovery of microbes

• Roman philosopher Lucretius (about 98– 55 B.C.) and the physician Girolamo Fracastoro (1478 –1553) suggested that disease was caused by invisible living creatures.

• The earliest microscopic observations made by the Italian Francesco Stelluti

The first person to observe and describe microorganisms accurately was the amateur microscopist Antony van Leeuwenhoek (1632 –1723) of Holland

His hobby was making microscopes…!!!

• Constructed simple microscopes composed of double convex glass lenses held between two silver plates • Could magnify around 50 to 270 times, • Illuminated his liquid specimens by placing them between two pieces of glass and shining light on them at a 45 ° angle to the specimen plane.

• A form of dark-field illumination • Bacteria clearly visible.

• Sent detailed letters describing his discoveries to the Royal Society of London.

•

Classification

: The orderly arrangement of units under study into groups of larger units.

• Nomenclature • Carolus Linnaeus, a Swedish botanist.

• Until 18 th century, the classification of living organisms placed all organisms into one of two kingdoms, plant and animal.

Kingdom Protista

• E. H. Haeckel, a German zoologist in 1866 • A third kingdom - Protista.

• To include those unicellular microorganisms that are typically neither plants nor animals.

– Bacteria, Algae, Fungi, and Protozoa – Viruses are not cellular – So, not classified as Protists

Kings Play Chess On Fine Glass Stools

– Domain – Kingdom – Phylum / Division – Class – Order – Family – Genus – Species

???

• What criteria could be used to distinguish a bacterium from a yeast or certain microscopic algae?

• Aid of Microscopy - observation of internal cell structure • In bacteria, the nuclear substance was not enclosed by a nuclear membrane.

• In algae and fungi, the nucleus was enclosed in a membrane.

• The presence / absence of membrane-bound internal structures is the criterion.

– Lower protists: Bacteria – Higher protists: Algae, Fungi, and Protozoa.

PROKARYOTES & EUKARYOTES

Groups Cell Wall FEATURE Size range Cell Membrane Location of Genetic content Nuclear Structure PROKARYOTES EUKARYOTES

Bacteria 1-2 by 1-4 μm Algae, Fungi, Protozoa, Plants and Animals Greater than 5 μm in width or diameter Absence of peptidoglycan Peptidoglycan (murein or mucopeptide) as component Generally do not contain sterols; contain part of respiratory and, in some, photosynthetic machinery Sterols present; Respiration and photosynthesis do not occur Nucleoid, chromatin body or nuclear material Not bounded by nuclear membrane; one circular chromosome Nucleus, mitochondria, chloroplasts Bounded by nuclear membrane; more than one chromosome Chromosome does not contain histones Nucleolus absent Chromosomes have histones Nucleolus present

Refer Pelczar, for a detailed list of differences.

Classification Systems

• • •

Two Kingdom System.......

–

Plantae / Animalia Five Kingdom System, 1969 Three Domain System, 1978

Whittaker’s Five-kingdom Concept

• A recent system of classification • Proposed by R. H. Whittaker (1969).

• Based on 3 levels of cellular organization that evolved to explain the 3 principal modes of nutrition: – photosynthesis, – absorption. and – ingestion.

• Kingdom Monera : – The Prokaryotes – They lack the ingestive mode of nutrition. • Kingdom Protista photosynthetic • • Kingdom Plantae • Kingdom Animalia absorptive Kingdom Fungi : – Unicellular eukaryotic microorganisms – All 3 nutritional types are represented here – The nutritional modes are continuous – The mode of nutrition of the microalgae is – The mode of nutrition of the protozoa is ingestive – The mode of nutrition in some other protists is

•

The Three Domains

–

Eukarya - all eukaryotic orgnaisms

»

Kingdoms:

» »

Animalia Plantae

» »

Fungi Protista

–

Bacteria

»

Bacteria, spirochetes, cyanobacteria etc

–

Archaea - "ancient" bacteria living extreme environments

»

methanogens

» »

extreme halophiles extreme thermophiles

Bergey’s Manual of Systematic Bacteriology The international standard for bacterial taxonomy • Kingdom Prokaryotae: All bacteria included • Divided into 4 divisions as follows: • Division 1: Gracilicutes – Prokaryotes with a complex cell-wall structure characteristic of Gram-negative bacteria • Division 2: Firmicutes – Prokaryotes with a cell-wall structure characteristic of Gram positive bacteria • Division 3: Tenericutes – Prokaryotes that lack a cell wall • Division 4: Mendosicutes – Prokaryotes of an earlier phylogenetic origin.

Viruses

• Very small noncellular obligate parasites or pathogens of plants, animals, and bacteria as well as other protists.

• So small that they can be visualized only by the electron microscope.

• Can be cultivated only in living cells.

• Range: 0.015 - 0.2 μm • Does not grow on artificial laboratory media • Electron microscopy required to see viruses

Bacteria

– Procaryotic – Unicellular procaryotic organisms or simple associations of similar cells. – Reproduction asexual: Cell multiplication is usually by binary fission.

– Grows on artificial laboratory media: characteristically by simple cell division – Range: 0.2 by 100 μm

Protozoa

– Unicellular eukaryotic organisms.

– Differentiated on the basis of morphological, nutritional, and physiological characteristics. – Range: 2 - 200 μm – Some are intracellular parasites – Reproduction by asexual & sexual processes

Fungi

– Eukaryotic lower plants devoid of chlorophyll.

– Usually multicellular but are not differentiated into roots, stems, and leaves.

– Single-celled microscopic yeasts to Molds to giant multicellular mushrooms and puffballs.

– Composed of filaments and masses of cells which make up the body of the organism, known as a mycelium.

– Reproduce by fission, budding, or by means of spores.

Yeasts

• Range: 5 -10 μm • Eukaryotic; unicellular.

• Reproduction by asexual cell division, budding, or sexual processes • Production of alcoholic beverages; also used as food supplements

Molds

• Range: 2 -10 μm by several mm • Eukaryotic; multicellular • Reproduction by asexual and sexual processes • Responsible for decomposition (deterioration) of many materials; • Useful for industrial production of penicillin

Algae

– Range: 1.0 μm to many feet – Eucaryotic; unicellular and multicellular: reproduction by asexual and sexual processes – Other algae, such as large brown Kelp, have a complex structure with cell types specialized for particular functions.

– All algae contain chlorophyll & are capable of photosynthesis.

–Found most commonly in aquatic environments or in damp soil.

–Important to the production of food in aquatic environments: –Used as food supplement and in pharmaceutical preparations –Source of agar for microbiological media; some produce toxins.

MICROSCOPE

• Roger Bacon (1220-1292) postulated that disease is produced by invisible living creatures.

• Girolamo Fracastoro (1483-1553) & Anton von Plenciz in 1762, but without proof.

• In 1658, Athanasius Kircher (1601-1680) referred to “worms” invisible to the naked eye in decaying bodies, meat, milk, and diarrheal secretions.

• In 1665, Robert Hooke’s description of cells in a piece of cork • Anton van Leeuwenhoek (1632 – 1723) was the first to report his observations with accurate descriptions and drawings.

• “

wee animalcules

” which we recognize as free-living protozoa.

SPONTANEOUS GENERATION vs BIOGENESIS

• Aristotle: “Animals might originate spontaneously from the soil, plants, or other unlike animals” • An accepted fact: Maggots could be produced by exposing meat to warmth and air.

• Francesco Redi (1626-1697) – Placed meat in a jar covered with gauze. – Attracted by the odor of the meat, flies laid eggs on the covering, and – From the eggs maggots developed. – Hence the experiment established the fact that the origin of the maggots was the flies and not the “meat”

• John Needham – Meat exposed to hot ashes, observed the appearance of organisms not present at the start of the experiment and concluded that the bacteria originated from the meat. • Lazaro Spallanzani – Boiled beef broth for 1 hour & then sealed flasks. – No microbes appeared following incubation.

• Needham argued that air was essential to the spontaneous production of microscopic beings and by sealing boiled infusions tubes • But still, support growth” …..

• Franz Schuize & Theodor Schwann – Schuize passed air through strong acid solutions into – Schwann passed air into his flasks through red-hot – In neither case did microbes appear. – “Acid and heat altered the air so that it would not

• In 1850, H. Schroder and T. von Dusch – Passed air through cotton into flasks containing heated broth.

– Thus the microbes were filtered out of the air by the cotton fibers so that, • Growth did not occur – A basic technique of plugging bacterial culture tubes with cotton stoppers was initiated.

A last attempt…..by Felix Archimede Pouchet, who published a report in 1859 “proving” Spontaneous Generation…  The final answer…  By the Father of Microbiology

• Louis Pasteur – Prepared a flask with a long, narrow gooseneck opening – Nutrient solutions were heated in the flask – Air - untreated and unfiltered - could pass in or out: but the germs settled in the gooseneck – No microbes appeared in the solution

A lasting assault…!!!

• In 1877, English physicist John Tyndall – Demonstrated that dust did indeed carry germs and – if dust was absent, broth remained sterile even if directly exposed to air.

THE GERM THEORY OF DISEASE

• Fracastoro of Verona suggested that diseases might be due to invisible organisms transmitted from one person to another • Von Plenciz stated that living agents are the cause of disease and suspected that different germs different diseases.

were responsible for

• Robert Koch (1843-1910) – The father of Medical Microbiology – Anthrax problem – Discovered the typical bacilli with squarish ends in the blood of cattle that had died of anthrax.

– Grew these bacteria in cultures in his laboratory – Examined them microscopically to be sure he had only one kind present

• Injected them into other animals • These became infected and developed clinical symptoms of anthrax.

• From these experimentally infected animals, he isolated microbes like those he had originally found in sheep that died of anthrax.

• The first time, a bacterium had been proved to be the cause of an animal disease.

• This series of establishment of …..

observations led to the

KOCH’s POSTULATES

Provided guidelines to identify the causative agent of an infectious disease.

• (1) A specific organism can always be found in association with a given disease.

• (2) The organism can be isolated and grown in pure culture in the laboratory.

• (3) The pure culture will produce the disease when inoculated into a susceptible animal.

• (4) It is possible to recover the organism in pure culture from the experimentally infected animal.

Pure Cultures

• The growth of a mass of cells of the same species in a laboratory vessel (such as a test tube) is called a pure culture.

• Laboratory procedures microorganisms to representing isolate each species and to grow (cultivate) each of the species separately.

Pasteur’s exptl demo of Koch’s Postulates

PUZZLED !!!???

After few weeks,

• Inoculated healthy chickens with his pure cultures, but chickens failed to die …!!!!

• Found that he had accidentally used cultures several weeks old • Repeated the experiment, using 2 groups of chickens.

– One group inoculated at the first demonstration with the old cultures that had proved ineffective, and – the second had not been previously exposed.

• Both groups received bacteria from fresh young cultures.

• First group remained healthy….!!!!

• Second group got sick and died, • Explanation: – Bacteria still retained their capacity for stimulating the host to produce substances, i.e.

antibodies, that protect against subsequent exposure to virulent organisms.

Edward Jenner

• To test his theory, drawn from folklore of the countryside – “Milkmaids who suffered the mild disease of cowpox never contracted smallpox” • In 1796, the famous experiment on 8-year-old James Phipps.

• Pus taken from a cowpox pustule from a dairymaid, on May 14, 1796.

• Injected it into an incision on the boy's arm.

• Subsequent inoculation with smallpox • Demonstrated the boy's immunity to the disease. • Jenner's first vaccination of a human patient.

Sarah Nelmes

, a milkmaid

• Proved that having been inoculated with cowpox, Phipps was immune to smallpox.

• Submitted a paper to the Royal Society in 1797 • Was told that his ideas were too revolutionary and that he needed more proof …!!!

• Experimented on more children, including his own 11-month-old son.

• In 1798, published results • Coined the word vaccine from the Latin 'vacca' for cow.

• Was widely ridiculed; Claimed it was “ungodly” to inoculate someone with material from a diseased animal.

• Soon, the advantages of vaccination and the protection it provided won out, and vaccination soon became widespread.

• The last endemic smallpox infection was recorded in 1977 • The last known case of death due to smallpox in 1978.

• The WHO declared the disease completely eradicated two years later.

Back on stage… Pasteur

• To make a vaccine for hydrophobia, or rabies, • A disease transmitted to people by bites of dogs, cats, etc..

• Joseph Meister bitten by a mad wolf.

• Disease could be produced in rabbits by inoculating them with saliva from mad dogs.

• Brain and spinal cord could be removed from the infected rabbits, dried, pulverized, and mixed with glycerin.

• Injected this mixture into dogs protected them against rabies • Also, Injected this mixture into Joseph Meister • Did not die…..!!!! Recovered