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Review of Lecture 1

-What is Microbiology?-Why do we study it?

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Today’s lecture Historical roots of Microbiology

• van Leeuwenhoek – microscopy • Pasteur – downfall of spontaneous generation• Koch – germ theory of disease, Koch’s postulates• Beijerinck – Enrichment culture and virology

1632-1723 1822-1895 1843-1910 1851-1931

Antonie van Leeuwenhoek (Holland, 1632-1723)

Antonie van Leeuwenhoek (1684)made microscopes and was the first to see bacteria in rain-water thatcollected in his garden and inpepper water. He recognized the common bacterial morphologies and accurate estimated their size.

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Leeuwenhoek’s observations

Leeuwenhoek documentedthe common bacterial morphologies and accuratelyestimated their sizes. He alsoobserved motile rods: they“swim about among one anothergently like a swarm of mosquitoes in the air…”

- de Kruif 1923

Microscopy

1. Light Microscopy – used to look at intact cells at low magnification i.e. bright field, phase contrast, dark field and fluorescence microscopy.

2. Electron Microscopy (EM) - used to look at internal structures or details of cell surfaces. i.e. Scanning (SEM) and Transmission (TEM)

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The Light Microscope

At full mag. an average bacterium appears 1mm

Magnification is calculated by multiplying the magnification due to the objective lens(10x, 40x, or 100x) by the magnification due to the ocular lens (10x).(1000x)(1µm)=1mm

Oil immersion

The 100x objective must be immersed in oil in

order to trap light necessary to see thespecimen.

oilair

slide

Cover slip

Objective lens

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The Problem of Contrast -Staining

Bacteria lacking pigments are difficult to see in the light microscope because of insufficientcontrast between water and the specimen.Staining cells with colored dyes increasescontrast but the cells are normally killed and often warped during the staining process.

The spontaneous generation controversy

Spontaneous generation is the theory that lifecan arise spontaneously from nonliving materials.

• Favored by Aristotle 3rd century BC• Francesco Redi (1626-1697) proved thatmaggots could not arise spontaneously fromdecaying meat.

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Microbes & spontaneous generation

What about Leeuwenhoek’s microbes?

• Lazarro Spallanzani (1729-1799) used seedinfusions as media and showed that hermeticallysealed flasks that were boiled for 1-2 hours remainedsterile. Nevertheless, cynics argued that boilingdestroyed the mysterious “vegetative force” or thatair elasticity was damaged by heating.

Pasteur & spontaneous generation

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Louis Pasteur (France, 1822-1895)Pasteur is considered father of appliedmicrobiology. Pasteur found that

bacteria – not yeast – were in the bad vats of wine and that he could kill the bacteria by heating the

vats after fermentation: Pasteurization.Pasteur also developed the generalprotocols for sterilizing media throughpressure and heat. He also found that

yeast require nitrogen to grow.

Modern Methods in Sterilization Stem from Pasteur

• An autoclave is a sealed device which heats steam to 121ºC at 15 lbs/in2. Moist heat kills cells more efficiently than dry heat.

• Pasteurization is a process whereby heat-sensitive food is heated to kill microbes that cause disease and food spoilage. In Pasteurization, the food is not necessarily sterilized.

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Pasteur & The Germ Theory of Disease

Pasteur was asked to study a disease of silk worms and found that globules – protozoa -inside sick moths grew as the disease progressed. He asserted that the parasites caused the disease.

The theory that microbes cause disease is called the Germ Theory of Disease. He saved France’s silk industry by breeding only healthy moths.

Robert Koch (Germany, 1843-1910)

Koch studied the cattle diseaseanthrax and proved without a doubt that it was caused by abacterium called Bacillusanthracis. He also pioneered the isolation of and study of pure cultures and invented the Petri dish and the use of solid media.

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Koch’s Postulates

1. The suspected pathogen is presentin all cases of the disease and absentfrom healthy animals.

2. The suspected pathogen should begrown in pure culture.

3. Cells from a pure culture shouldcause the disease in healthy animals.

4. The suspected pathogen should bereisolated and shown to be the sameas the origninal.

Pasteur & Koch fight disease

Pasteur developed vaccines based onweakened, or attenuated, microbes for…• Chicken cholera• Anthrax• RabiesKoch proved that tuberculosis was causedby a bacterium, Mycobacterium tuberculosis

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Complex Medium

Nutrient BrothNutrient Broth• Peptone from meat 4.3 g/l• Sodium chloride 6.4 g/l• Peptone from casein 4.3 g/l• H20 to 1 liter• Final pH (37 °) 7.5

Defined Medium

Modified Medium B• (NH4)2SO4 0.25 g/l• Glucose 0.25 g/l• Na2HPO4 0.071 g/l• Modified Hutner’s Basal Salts (20 ml)• H20 to 1 liter

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Aseptic Technique

Aseptic Technique

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Aseptic Technique – Streak Plate

1.

2.

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Isolation of a Pure Culture -Spread Plates & Pour Plates

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Martinus Beijerinck(1851-1931)

Beijerinck pioneered the enrichment culture. In an enrichment culture, the scientist selects bacteria with specific physiological properties to grow by designing media with specific formulations. Using enrichments, he was able to isolate nitrogen-fixing bacteria and many other bacteria. He also described filterable infectious agents – viruses.

Macronutrients

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Trace elements

Growth factors & Vitamins

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Terms related to cultivationA medium(a) is a nutrient solution used togrow microorganisms.

Sterilization is the process of killing all microbes in or on objects.

A pure culture is a culture containing a singlekind of microbe.

When a microbe grows to a density such that amedium is cloudy it is said to be turbid.

An overview of microbial life

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Outline

• Cell structure• Microbial diversity• Cells vs viruses• Tree of life• Major groups of microorganisms

2.1 Elements of cell and viral structure• Membrane defines it: nutrients/waste• Cytoplasm: contains macromolecules and other substances• Cell wall support to cell (microbes and plants)• Eukaryotes- organelles: algae, fungi, protozoa and

metazoans• Prokaryotes- small (!m range), simpler, Bacteria and

Archea

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Viruses• Not cells: non-dynamic, need host and cell machinery to reproduce,

contain genes• No ribosomes, no metabolic activities• Infect cells and can be used as genetic tool• Size: nm range

2.2 Arrangement of DNA in microbial cells• DNA: coding materialEukaryotes (nucleus) Prokaryotes (nucleoid)Linear molecules in pairs (diploid) Bacterial chromosome(chromosome) number ranges single copy, circularComplex with proteins plasmids containMitosis-duplicates nonessential genesMeiosis- gamete formation (haploid)

• GenesNumber varies in spp- E. coli (4,300) human (1000x)E. coli proteins 1,900 and 2.4 million total-expression

Gene expression- characteristic of eukaryotes and prokaryotes (differences among species)

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2.3 Tree of life• Phylogenetic analysis

– Extraction– Common gene is amplified– Sequence– Sequence are aligned– Tree making

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2.4 Physiological diversity

• Energy and Carbon

Carbon

• Heterotrophs: organic compounds as their source

• Autotrophs: CO2 (atmosphere)

– Chemoorganotrophs are hetereotrophs– Chemolithotrophs/phototrophs are autotrophs

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Extremes

• Metabolic diversity allows exploitation of very diverse environments (extreme)

• Human ideal pH7 and 25C• Extremophiles (require)

– Temperature: hyperthermophile, psycrophile0-113 C– pH: acido- or alkali- phile (4, 12)– Pressure: Barophile (1000 atm)– Salt:halophile (32%)

2.5 Diversity

Bacteria

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Gram+

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Archaea Hyper thermoMethanogensAcidophilesHalophiles

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Thermoplasma

Eukaryotes

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lichens