What is Food Microbiology?

The focus of Food Microbiology is on the detection and analysis of foodborne spoilage microorganisms.

Food microbiology is the study of food micro-organisms; how we can identify and culture them, how they live, how some infect and cause disease and how we can make use of their activities.

Microbes are single-cell organisms so tiny that millions can fit into the eye of a needle.

They are the oldest form of life on earth. Microbe fossils date back more than 3.5 billion years to a time when the Earth was covered with oceans that regularly reached the boiling point, hundreds of millions of years before dinosaurs roamed the earth.

The field of food microbiology is a very broad one, encompassing the study of microorganisms which have both beneficial and deleterious effects on the quality and safety of raw and processed foods.

Food science is a discipline concerned with all aspects of food - beginning after harvesting, and ending with consumption by the consumer. It is considered one of the agricultural sciences, and it is a field which is entirely distinct from the field of nutrition. In the U.S., food science is typically studied at land-grant universities.

Examples of the activities of food scientists include the development of new food products, design of processes to produce these foods, choice of packaging materials, shelf-life studies, sensory evaluation of the product with potential consumers, microbiological and chemical testing. Food scientists in universities may study more fundamental phenomena that are directly linked to the production of a particular food product.

Food scientists are generally not directly involved with the creation of genetically modified (bio-engineered) foods.

Some of the subdisciplines of food science: Food safety, Food engineering, Product development, Sensory analysis, Food chemistry.

The primary tool of microbiologists is the ability to identify and quantitate food-borne microorganisms; however, the inherent inaccuracies in enumeration processess, and the natural variation found in all bacterial populations complicate the microbiologists job.

Without microbes, we couldn’t eat or breathe.

Without us, they’d probably be just fine.

Understanding microbes is vital to understanding the past and the future of ourselves and our planet.

Archaea look and act a lot like bacteria. So much so that until the late 1970s, scientists assumed they were a kind of “weird” bacteria.

Then microbiologist Carl Woese devised an ingenious method of comparing genetic information showing that they could not rightly be called bacteria at all. Their genetic recipe is too different.

So different Woese decided they deserved their own special branch on the great family tree of life, a branch he dubbed the Archaea.

Archaea comes from the Greek word meaning “ancient.” An appropriate name, because many archaea thrive in conditions mimicking those found more than 3.5 billion years ago. Back then, the earth was still covered by oceans that regularly reached the boiling point — an extreme condition not unlike the hydrothermal vents and sulfuric waters where archaea are found today.

Some scientists consider archaea living fossils that may provide hints about what the earliest life forms on Earth were like, and how life evolved on our planet.

In addition to superheated waters, archaea have been found in acid-laden streams around old mines, in frigid Antarctic ice and in the super-salty waters of the Dead Sea. A number of other extreme-living bacterial species also enjoy these conditions, too, such as the community of cyanobacteria and bacteria shown top right.

Foodborne illness or food poisoning is caused by consuming food contaminated with pathogenic bacteria, toxins, viruses, prions or parasites. Such contamination usually arises from improper handling, preparation or storage of food. Foodborne illness can also be caused by adding pesticides or medicines to food, or by accidentally consuming naturally poisonous substances like poisonous mushrooms or reef fish. Contact between food and pests, especially flies, rodents and cockroaches, is a further cause of contamination of food.

Some common diseases are occasionally foodborne mainly through the water vector, even though they are usually transmitted by other routes. These include infections caused by Shigella, Hepatitis A, and the parasites Giardia lamblia and Cryptosporidium parvum.

• Thermophiles like unusually hot temperatures. A few species have been found to survive even above 110 degrees Celsius (water boils at 100 degrees Celsius). • Psychrophiles like extremely cold temperatures (even down to -10 degrees Celsius). • Halophiles thrive in unusually salty habitats. Some can thrive in water that’s 9% salt; sea water contains only 0.9% salt. • Acidophiles prefer acidic conditions; Alkaliphiles prefer very alkaline environs.

Accumulating sufficient data on the behaviour of microorganisms in foods requires an extensive amount of work, and is costly. In addition, while data alone can describe the response of a microorganisms in food, they provides little insight into the relationship between physiological processes and growth or survival.

Scientists also use molecular tools to extract and compare bits of a particular kind of RNA from samples in order to determine if previously known or new microbes are present in a particular environment. This technique is widely used as a biomarker and for microbial ecology studies. It uses a particular kind of RNA known as 16S ribosomal RNA, or 16S rRNA.

Ribosomes are the gene-translating machines in all living things. When a gene on a piece of DNA is copied into a strand of messenger RNA and ferried out of the cell nucleus into the cell fluid, ribosomes there latch onto this mRNA. The ribosomes move along the mRNA strand, reading the code contained in its sequence of nucleotide bases (the As, Gs, Cs and Us, since U replaces T in RNA) and stringing the right amino acids together based on the code to build protein chains.

Pasteurization is typically associated with milk. There are two widely used methods to pasteurize milk: high temperature/short time (HTST), and ultra-high temperature (UHT). HTST is by far the most common method. Milk simply labelled "pasteurized" is usually treated with the HTST method, whereas milk labelled "ultra-pasteurized" must be treated with the UHT method. HTST involves holding the milk at a temperature of 161.5 degrees F (or 72 C) for at least 15 seconds. UHT involves holding the milk at a temperature of 280 degrees F (or 138 C) for at least two seconds.

Pasteurization methods are usually standardized and controlled by national food safety agencies (such as the USDA in the United States and the Food Standards Agency in the U.K.). These agencies require milk to be HTST pasteurized in order to qualify for the "pasteurized" label. There are different standards for different dairy products, depending on the fat content and the intended usage. For example, the pasteurization standards for cream differ from the standards for fluid milk, and the standards for pasteurizing cheese are designed to preserve the phosphatase enzyme, which aids in curing the cheese.

The HTST pasteurization standard was designed to achieve a 5-log (approximately one million-fold) reduction in the number of viable microorganisms in milk. This is considered adequate for destroying almost all yeasts, mold, and common spoilage bacteria and also to ensure adequate destruction of common pathogenic heat-resistant organisms (including particularly Mycobacterium tuberculosis, which causes tuberculosis and Coxiella burnetii, which causes Q fever). HTST pasteurization processes must be designed so that the milk is heated evenly, and no part of the milk is subject to a shorter time or a lower temperature.

HTST pasteurized milk typically has a refrigerated shelf life of two to three weeks, whereas ultra pasteurized milk can last much longer when refrigerated, sometimes two to three months. When UHT pasteurization is combined with sterile handling and container technology, it can even be stored unrefrigerated for long periods of time.

The genes for ribosomal RNA have changed little over millions of years as organisms evolved. The slight changes that have occurred provide clues as to how closely or distantly various organisms are related.

Because the 16S rRNA gene is very short, just 1,542 nucleotide bases, it can be quickly and cheaply copied and sequenced. So when a scientist has a test tube full of pond water or dirt from an arid mountainside, she must first pull out the rRNA that’s mixed up with all the other RNA, DNA and other stuff in that tube. To do this, she cleans and purifies the sample first, getting rid of unwanted debris.

She then uses one or several techniques designed to break open cells like a kid cracking open a piggybank. Now she has to find the 16S rRNA genes in and amongst all the other genes. Although 16S rRNA genes from different microbes will have a few different nucleotides scattered throughout the sequences, those nucleotides at the very beginning or end of the gene are the same from organism to organism.

The scientist uses several copies of another bit of RNA called a primer. A primer is like a mirror image of a short bit of RNA or single strand of DNA; that is, its sequence of nucleotides is the direct complement to the sequence of nucleotides in a known part of the target RNA or DNA.

In this instance, the primer would be the mirror image of the beginning or end of the 16S rRNA sequence. Because complementary nucleotides pair up like the two halves of Velcro, the primer enables the scientist to pick out the 16S rRNA in the sample. The scientist then uses PCR to make millions of copies of these genes. She then has enough 16S rRNA to compare the sequences of the genes from her sample to libraries of stored 16S rRNA genes from numerous known bacteria.

If some of her gene sequences match up perfectly, she knows that these are microbes that have been previously identified. But if others among her sampled sequences show differences, she knows she has found previously unknown microbes.

Bacteria can be found virtually everywhere. They are in the air, the soil, and water, and in and on plants and animals, including us. A single teaspoon of topsoil contains about a billion bacterial cells (and about 120,000 fungal cells and some 25,000 algal cells). The human mouth is home to more than 500 species of bacteria.

Some bacteria (along with archaea) thrive in the most forbidding, uninviting places on Earth, from nearly-boiling hot springs to super-chilled Antarctic lakes buried under sheets of ice. Microbes that dwell in these extreme habitats are aptly called extremophiles.

Like dinosaurs, bacteria left behind fossils. The big difference is that it takes a microscope to see them. And they are older.

Bacteria and their microbial cousins the archaea were the earliest forms of life on Earth. And may have played a role in shaping our planet into one that could support the larger life forms we know today by developing photosynthesis.

Cyanobacteria fossils date back more than 3 billion years. These photosynthetic bacteria paved the way for today's algae and plants. Cyanobacteria grow in the water, where they produce much of the oxygen that we breathe. Once considered a form of algae, they are also known as blue-green algae.

The human body consists of millions of different cells. A bacterium consists of a single cell.

A bacterium’s genetic information is contained in a single DNA molecule suspended in a jelly-like substance called cytoplasm. In most cases, this and other cell parts are surrounded by a flexible membrane that is itself surrounded by a tough, rigid cell wall. A few species, such as the mycoplasmas, don’t have cell walls.

Even though bacteria have only one cell each, they come in a wide range of shapes, sizes, and colors.

Does a bacterium’s cell wall, shape, way of moving, and environment really matter?

Yes! The more we know about bacteria, the more we are able to figure out how to make microbes work for us or stop dangerous ones from causing serious harm. And, for those of us who like to ponder more philosophical questions like the origins of the Earth, there may be some clues there as well.

Whether a bacteria has a thin or a thick cell wall determines what antibiotic will work against it. If you’ve ever been sick and waited for the results of a culture and sensitivity test, you may have heard the terms “Gram-positive” or “Gram-negative.”

Bacteria with thick cell walls retain dye from a cell-staining method developed by Christian Gram; bacteria with thin walls do not. Knowing the difference can and does save lives, time and money, and ensures that you or your loved one is getting the best and most effective treatment.

Some bacteria look like little balls (Micrococcus) while others appear like tangled strings or corkscrews (Leptospira) under a microscope. Others look like medicine capsules (Salmonella) or segmented ribbons (Cyanobacteria) or sticks. Still others look like fat commas (Vibrios).

Some bacteria are stalked (Caulobacter) while others have buds (Rhodomicrobium). Some have sheaths (Sphaerotilus) while others don’t.

Bacteria like mycoplasmas that lack a hard cell wall don’t have any particular shape at all.

Just like in animals, where size ranges from the giant blue whale to the tiny gnat, bacteria vary from 1 millimeter in diameter at the largest end of the scale to 20 nanometers in length at the smallest.

The largest bacteria found so far can actually be seen without the use of a microscope (Thiomargarita namibiensis and Epulopiscium fischelsoni). The smallest known bacteria are so tiny that they were once thought to be viruses (Mycoplasmas).

Some bacteria have hair- or whip-like appendages called flagella used to ‘swim’ around. Others produce thick coats of slime and ‘glide’ about. Some stick out thin, rigid spikes called fimbriae to help hold them to surfaces. Some contain little particles of minerals that orient with the planet’s magnetic fields to help the bacteria figure out whether they’re swimming up or down.

Fungi are eukaryotic organisms. This means that their DNA-containing chromosomes are enclosed within a nucleus inside their cells. (The chromosomes of bacteria and archaea are not walled off inside nuclei, making them prokaryotic organisms.)

Many decades ago, scientists thought that fungi were primitive kinds of plants. New studies looking at the DNA of fungi have confirmed that these organisms are not plants.

Unlike plants, fungi do not make their own food energy via photosynthesis, but dine on organic matter like rotting leaves, wood, and other debris, or upon the tissues of living plants and animals.

Fungi, along with bacteria, are the planet’s major composters and recyclers.

In addition to the standard HTST and UHT pasteurization standards, there are other lesser-known pasteurization techniques. The first technique, called "batch pasteurization", involves heating large batches of milk to a lower temperature, typically 155 degrees fahrenheit (or 68 C). The other technique is called higher-heat/shorter time (HHST), and it lies somewhere between HTST and UHT in terms of time and temperature.

The batch pasteurization step, which is cheap at a large scale, is often performed prior to standard pasteurization. Batch pasteurized milk is often called "raw milk" or, confusingly, "unpasteurized milk". It cannot be called "pasteurized", even though a significant number of pathogens are destroyed during the process.

In recent years, there has been some consumer interest in raw milk products, due to perceived health benefits. Advocates of raw milk maintain, correctly, that vitamins and nutrients survive much better in milk that has not been pasteurized. They also maintain that organic raw milk (most retail raw milk is also organic) is less likely to contain harmful pathogens due to better husbandry in organic dairy herds. This may be true, but it has not been proven.

However, doctors (and even most raw milk advocates) acknowledge that certain people (e.g. pregnant or breast-feeding mothers, those undergoing immunosuppression treatment for cancer, organ transplant or autoimmune diseases, and those who are immunocompromised due to diseases like AIDS) should not risk consumption of raw milk.

In fact, some doctors suggest that babies and breast-feeding mothers avoid all but UHT pasteurized dairy products.

In Africa, it is common to boil milk whenever it is harvested. This intense heating greatly changes the flavor of milk, which the people in Africa are accustomed to.

Although fungi may seem like a nuisance when they dine in your fruit bowl or refrigerator, their ability to degrade some of the toughest organic materials, including tree wood and insect exoskeletons, means that our planet is not cluttered with a mass of debris.

Fungi dine at home, eating whatever they’re growing on. Fungi secrete digestive enzymes in order to break down complex food sources, such as animal corpses and tree stumps, into smaller components they can absorb.

Algae are plant-like microorganisms that preceded plants in developing photosynthesis, the ability to turn sunlight into energy. Algae cells contain light-absorbing chloroplasts and produce oxygen through photosynthesis.

Although plants generally get the credit for producing the oxygen we breathe, some 75% or more of the oxygen in the planet’s atmosphere is actually produced by photosynthetic algae and cyanobacteria.

Algae also play an important role as the foundation for the aquatic food chain. All higher aquatic life forms depend either directly or indirectly on microscopic gardens of algae.

Most unicellular algae live in water, some dwell in moist soil, and others join with fungi to form lichens.

Green algae The most clearly plant-like algae, this species gets its namesake hue from high levels of chlorophyll.

Their cell walls are made up of cellulose, the same material that makes up the cell walls in larger, multicellular plants. Like plants, they store the food they make through photosynthesis as starches. Growing in large masses, these algae can form visible layers of slick, green scum on the surfaces and sides of ponds, puddles or damp soil.

Fossil records suggest that the first green algae originated 500 to 600 million years ago. Early algae probably gave rise to multicellular plants.

Dinoflagellates have long whip-like structures called flagella that let them turn, maneuver and spin about through the water. About 90% of these algae dwell in the ocean.

Some species glow in the dark in a process called bioluminescence. These species contain a compound called luciferin (the same compound found in fireflies). The glow increases markedly if the algae cells are agitated, as when a ship churns through the water.

About half the species of dinoflagellates are photosynthetic; the other half are predators that attack bacteria, algae, and even fish.

Dinoflagellate neurotoxins can concentrate in the bodies of shellfish and fish that eat the algal cells, in turn causing people who eat these seafoods to come down with illnesses such as paralytic shellfish poisoning and ciguatera (a combination of gastrointestinal, neurological, and cardiovascular disorders.)

So-called “red tides” occur when enormous blooms of trillions of dinoflagellates are triggered by an upwelling of nutrients from the water’s depths during warmer seasons. The population of dinoflagellates can jump to more than 20 million cells per liter of sea water along some coasts during these blooms, turning the water a reddish hue.

The name protozoa means “first animals.” As the principal hunters and grazers of the microbial world, protozoa play a key role in maintaining the balance of bacterial, algal, and other microbial life. They also are themselves an important food source for larger creatures and the basis of many food chains.

Protozoa have been found in almost every kind of soil environment from peat bogs to arid desert sands. They teem in the deep sea as well as near the surface of waters, and can be found even in frigid Arctic and Antarctic waters.

Some species of protozoa are part of the normal microbial flora of animals, and live in the guts of insects and mammals, helping to break down complex food particles into simpler molecules. A very small number of species cause disease in people, including Plasmodium vivax, which causes malaria.

Bacterial infection is the most common cause of food poisoning. In the United Kingdom during 2000 the individual bacteria involved were as follows: Campylobacter jejuni 77.3%, Salmonella 20.9%, Escherichia coli O157:H7 1.4%, and all others less than 0.1% [4] (http://www.food.gov.uk/science/sciencetopics/microbiology/58736).

Symptoms for bacterial infections are delayed because the bacteria need time to multiply. They are usually not seen until 12-36 hours after eating contaminated food.

Common bacterial foodborne pathogens are:

Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria Bacillus cereus Brucella spp. Campylobacter jejuni which causes Guillain-Barré syndrome Corynebacterium ulcerans Coxiella burnetii or Q fever Crohn's disease Escherichia coli O157:H7 enterohemorrhagic (EHEC) which causes hemolytic-uremic syndrome Escherichia coli - enteroinvasive (EIEC) Escherichia coli - enteropathogenic (EPEC) Escherichia coli - enterotoxigenic (ETEC) Listeria monocytogenes Plesiomonas shigelloides Salmonella spp. Shigella spp. Streptococcus Vibrio cholerae, including O1 and non-O1 Vibrio parahaemolyticus Vibrio vulnificus Yersinia enterocolitica and Yersinia pseudotuberculosis

The four main subgroups of protozoa are the ciliates, the flagellates, the sarcodina, and the apicomplexans.

A few ciliates can grow up to 2 millimeters in length, big enough to be seen without a microscope.

Flagellates Similarly complex single-celled organisms, flagellates have whip-like appendages called flagella sticking out of their cells.

The flagella are used for locomotion and to direct food particles or cells into the organism’s mouth-like opening. Flagellates dine on bacteria, algae, and other protozoa.

Several well-known flagellates cause parasitic diseases, such as trypanosomes that cause sleeping sickness, and Giardia lamblia, a parasite found in mountain streams and rivers that causes severe gastrointestinal distress.

Heliozoa, radiozoa, and forams tend to be passive grazers and predators, relying on suitable food swimming or drifting past to come into contact with their pseudopods.

Several species in the sarcodina group, including some species of amoebas, cover themselves with protective shell-like coverings called tests. These tests are stippled with many small and large openings through which water can flow in and out and through which the pseudopods protrude.

The tests of radiozoa are made up of silica (the same substance in diatom cell walls) and can form very intricate, lacy designs that may be studded with long spines that increase buoyancy and ward off predators.

The tests of forams are made up of sand grains or organic compounds. These can become quite large, the biggest reaching a little over 2 inches in diameter, making them some of the largest single-celled organisms known.

When forams die, their tests sink and accumulate in large batches; the Great Pyramids of Egypt are built from sandstone composed largely of fossilized giant Nummulites, an ancient kind of foram. The famous White Cliffs of Dover are limestone cliffs formed from the skeletal remains of forams.

Apicomplexans These protozoa are obligatory intracellular parasites: they must spend at least part if not all of their life cycle in a host animal.

Apicomplexans are characterized by the presence of special organelles (tiny organ-like structures) located at the tips (apices) of the cells. These organelles contain enzymes that punch through, slice open and otherwise penetrate host tissues.

The best known apicomplexan is Plasmodium, the agent that causes malaria. Plasmodium spends part of its life cycle in mosquitoes and the other part in human hosts where it ultimately infects and ruptures blood cells in large numbers.

Another familiar apicomplexan is Cryptosporidium parvum. This water-borne parasite forms extremely durable cyst-like structures that enable it to survive UV radiation and sometimes chlorine in swimming pools and treated water. An outbreak of Cryptosporidium in Milwaukee’s drinking water supply in 1993 killed 50 people and sickened more than 400,000.

Probably the best known and most deadly case of contamination in the U.S. in recent years happened in Milwaukee in 1993, when the municipal water supply was contaminated by Cryptosporidium, an intestinal protozoan. At least 50 people died, and some 400,000 people became ill, 4,000 badly enough to be hospitalized.

In 1973, a Dallas resident went out to the backyard only to stumble upon a reddish, jelly-like mass pulsating in the grass. News reports on the discovery claimed that a “new life form” had been found, and many people couldn’t help recalling the cult classic sci-fi thriller The Blob.

Scientists called to the scene, however, put any fears of menacing goo or alien creatures to rest by identifying the mass as an unusually large (46 centimeters or more than 14 inches in diameter) plasmodial slime mold.

Slime molds were once considered fungi, but unlike fungi, they can move, and their cell membranes are made of different stuff.

Slime molds are made up of individual cells that form an aggregate mass. In their visible, aggregate states, they look like blobs, gooey or foamy masses, spilled jelly, or even dog vomit. They may be bright orange, red, yellow, brown, black, blue, or white.

These large masses act like giant amoebas, creeping slowly along and engulfing food particles along the way. If a slime mold aggregate is diced up, the pieces will pull themselves back together. The blobs can navigate and avoid obstacles and if a food source is placed nearby, they seem to sense it and head unerringly for it. j, a. There are two kinds of slime molds. Plasmodial slime molds (the most common kind) share one big cell wall that surrounds thousands or millions of nuclei. Proteins called microfilaments act like tiny muscles that enable the mass to crawl at rates of about 1/25th of an inch per hour.

As long as there is enough food and moisture, the mass thrives. But when food and water are scarce, the mass separates into smaller blobs. The Plasmodium forms stalks topped by sphere-like fruiting bodies that contain spores that are carried by the rain or wind to new locations.

Cellular slime molds also produce spores, but these germinate into amoeba-like cells. The cells happily go their individual ways, as long as food and water are available.

When nutrients and moisture are scarce, individual cells send out a chemical beacon to attract other cells of the same species. The cells join up to form a mass that looks and acts like a slug to take them to a more favorable location.

Cells in cellular slime molds retain their individual cell walls when they form a mass, so the visible slug is actually a collection of hundreds of thousands of individual cells joined together.

Slime molds eat decaying vegetation, bacteria, fungi, and even other slime molds. They are most commonly found in forests.

Milk pasteurization standards have been subject to increasing scrutiny in recent years, due to the discovery of pathogens that are both widespread and heat resistant (able to survive pasteurization in significant numbers). Researchers have developed more sensitive diagnostics, such as real-time PCR and improved culture methods, that have enabled them to identify pathogens in pasteurized milk.

Note: The following paragraphs in this section discuss controversial, ongoing research.

One bacterium in particular, the organism Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne's disease in cattle and is suspected of causing at least some Crohn's disease in humans, has been found to survive pasteurization in retail milk in the U.S., the U.K., Greece, and the Czech Republic. The food safety authorities in the U.K. have decide to re-evaluate pasteurization standards in light of the MAP results and other evidence of harmful, pasteurization-resistant pathogens.

The USDA (which is responsible for setting pasteurization stardards in the U.S.) has not re-evaluated their position on pasteurization adequacy. They do not dispute the studies, which are at this point accepted by the scientific community, but maintain that the presence of MAP in retail pasteurized milk must be due to post-pasteurization contamination. However, some researchers within the FDA, which is responsible for food safety in the U.S., have begun pushing for a re-evaluation of these results. There is a small but growing body of criticism directed at these agencies by Crohn's disease sufferers, scientists, and doctors. Some have suggested that the U.S. dairy industry has been successful in suppressing the agencies' response to a potential health crisis, for fear of consumer panic which would lead to a decrease in milk consumption. It is worth noting that while MAP has not been definitely proven to be harmful in humans, all other mycobacteria are pathogenic, and it has been definitively shown to cause disease in cattle and other ruminants.

The term cold pasteurization is used sometimes for the use of radioactivity or other means (e.g. chemical) to kill bacteria in food.

Water molds are always found in wet environments, especially in fresh water sources and near the upper layers of moist soil.

Officially named Oomycota, they are also known as downy mildews and white rusts.

Water molds were long considered fungi because they produce fungi-like filamentous hyphae and feed on decaying tissue like rotting logs and mulch.

The Oomycota species Phytophthora infestans caused the Great Potato Famine that killed nearly a million people in Ireland in 1846–1847. The water mold virtually wiped out the country’s potato crops, which were an essential staple in the Irish diet (sometimes the only food on the table.)

Microbes break down food molecules our body’s enzymes and acids can’t dissolve, helping us squeeze all the nutrients out of our food. Some make valuable vitamins that our body needs. Many microbial species have proved to be consummate evolutionary wheelers and dealers, arranging collaborations, mergers, and acquisitions that usually serve both partners well. If you could peer deep into one of the many cells in your body, you’d see little blobs, squiggles, and coils. e, k. These are the cell’s organelles, structures that perform specialized functions in cells the same way that the lungs, heart, and other organs do in a body.

Mitochondria are the energy factories found in each cell of fungi, protozoa, insects, and animals. Once nutrients are absorbed or digested, they move in the form of minuscule molecules into the mitochondria, which convert the molecules into chemical energy to power the cell.

Chloroplasts undertake a similar function in the cells of plants, algae, and some protozoa. They capture sunlight and, through a series of chemical reactions called photosynthesis, use the light to make energy.

These organelles are absolutely essential to the existence of all higher life forms on Earth. If all of the mitochondria in our bodies were to suddenly shut down, we would die. The same is true for plants were they to lose their chloroplasts.

So where did they come from?

As microscopes improved over the years, scientists began noticing striking similarities in the appearances of mitochondria, chloroplasts, and bacterial cells. They discovered that these two organelles contain their own DNA, or gene set, organized very much like the DNA in bacterial cells.

Mitochondria and chloroplasts also reproduce independently from the cells in which they reside, in a manner very like bacterial fission.

Many microbiologists think it is likely that mitochondria and chloroplasts were once free-living prokaryotes (cells that lack a nucleus and organelles) that somehow took up residence in larger cells.

An Invading bacterium may have infected a cell, then become a permanent resident as it adapted to become less virulent, or the target cell became less susceptible. Or both.

Chloroplasts likely first entered a host cell as food before establishing a successful merger with the cell so they were not digested.

Ages ago, as land plants were evolving, they ran into a few impediments. Soil can sometimes prove a nutrient-poor and inhospitable environment. In order to grow and thrive, plants need nitrogen to make proteins, but they lack the chemistry set to convert free nitrogen in the air into a form their cells can use. l, e, d, l, i. To overcome these obstacles, early plants struck deals with co-evolving bacteria and fungi.

Some early bacteria developed the chemical tools to harvest free nitrogen from the air and convert it into forms such as ammonia and nitrate through a process called nitrogen fixation. The catch is that they need sufficient amounts of energy in the form of carbohydrates to power these conversions, and the supply of carbohydrates in the soil can be limited.

On the other hand, plants produce copious amounts of carbohydrates as a product of photosynthesis. And ammonia and nitrate are perfect protein-building nitrogen forms for plants.

The bacteria moved into the plants’ roots, forming bumps on the roots called nodules that supply the fixed nitrogen plants need. In exchange, the plants supply the bacteria with the carbohydrates they require. Because Rhizobia can still dwell independently in the soil, plants are more dependent upon them than the microbes are on plants.

The prevention is mainly the role of the state, through the definition of strict rules of hygiene and a public service of veterinary survey of the food chain, from farming to the transformation industry and the delivery (shops and restaurants). This regulation includes:

traceability: in a final product, it must be possible to know the origin of the ingredients (originating farm, identification of the harvesting or of the animal) and where and when it was processed; the origin of the illness can thus be tracked and solved (and possibly penalized), and the final products can be removed from the sale if a problem is detected; respect of hygiene procedures like HACCP and the "cold chain"; power of control and of law enforcement of the veterinarians. At home, the prevention mainly consists of:

the respect of the food storage and food preservation methods (especially refrigeration), and checking the expiration date; washing the hands before preparing the meal and before eating; washing the fresh vegetables with clear water, especially when not cooked (e.g. fruits, salads); washing the dishes after use; keeping the kitchen clean. Bacteria need warmth, moisture, food and time to grow. The presence, or absence, of oxygen, salt, sugar and acidity are also important factors for growth. In the right conditions, one bacterium can multiply using binary fission to become four million in eight hours. Since bacteria can be neither smelled nor seen, the best way to ensure that food is safe is to follow principles of good food hygiene. This includes not allowing raw or partially cooked food to touch dishes, utensils, hands or work surfaces previously used to handle even properly cooked or ready to eat food.

High salt, high sugar or high acid levels keep bacteria from growing, which is why salted meats, jam, and pickled vegetables are traditional preserved foods.

The most frequent causes of bacterial foodborne illness are cross-contamination and inadequate temperature control. Therefore control of these two matters is especially important.

Thoroughly cooking food until it is piping hot, i.e. above 70°C (158°F) will quickly kill virtually all bacteria, parasites or viruses, except for Clostridium botulinum and Clostridium perfringens, which produces a heat-resistant spore that survives temperatures up to 100°C (212°F). Once cooked, hot foods should be kept hot - above 63°C (145°F) stops microbial growth.

Cold foods should be kept cold, below 5°C (41°F). However, Listeria monocytogenes and Yersinia enterocolitica can both grow at refrigerator temperatures.

Animals need nitrogen for protein building, too. We humans get our nitrogen by eating plants (or by eating animals that eat plants).

Another partnership teams plants with soil-dwelling fungi called mycorrhizae. Virtually all plants from flowers to towering trees like Sequoias have partner mycorrhizae.

Some species of mycorrhizae cover the surface of plants’ root hairs; others settle down inside the plant roots. The fungi act as extensions of the plants’ roots, vastly increasing the surface space of their nutrient-absorbing network.

Mycorrhizae increase the plants’ uptake of water and essential nutrients, particularly phosphorous, which doesn’t spread readily in soil. In exchange, the plants provide the fungi energy in the form of carbohydrates. This partnership enables both plants and fungi to survive in nutrient-poor places where they otherwise might die.

Major development projects are taking place in oceans across the globe all the time, enterprises that will provide shelter and food for a vast number of fish, mussels, urchins, and other marine life.

While credit is regularly and duly given to the visible construction crew — coral polyps — recognition is also due the polyps’ invisible, but very active algal partners, the zooxanthelle.

These algae (a type called dinoflagellates) live inside the body tissues of coral polyps. Coral polyps take care of some of their nutritional needs on their own by catching tiny protists and organic matter that drift past their tentacles. Their partnership with the chlorophyll-containing algae enable them to also get food from sunlight as if they were plants.

The zooxanthelle do the actual work of converting the sunlight into energy via photosynthesis. The by-products they generate (organic carbons such as glycerol and sugars) are excellent nutrients for their polyp hosts.

The zooxanthellae supply much of the polyps’ energy needs. In turn, the polyps provide the algae a protected, stable environment and nutrients they need for growth, such as nitrates and phosphates. Zooxanthellae also increase the rate of coral calcification, the growth of the hard shells that form the actual reef structures.

Ants lack the necessary enzymes to digest leaves and stems themselves, so they cut them up and feed them to the fungi, which break down cellulose (the tough fibrous material in plant tissues), making nutrients available to the ants.

The ants excavate nests and build nice, cozy, safe chambers for their fungal gardens; they clean off debris and parasites from the fungi; and even produce special antibiotics in their bodies to ward off or kill infectious organisms that might attack their crops.

In return, the fungi produce swellings at the tips of their hyphae (long strings of fungal cells) that are rich in proteins, sugars and other nutrients. The ants dine on these nutritious swellings, called gongylidia.

The fungi are ensured a copious food supply and a stable, nurturing environment in which to live.

The fungi rely upon the ants for reproduction. Before new queen ants fly off to mate and found their own colonies, they tuck a bit of fungus in their mandibles to start their new gardens. The fungi growing in virtually every leaf-cutter garden are actually clones of the same fungus farmed by ants 25 million years ago.

Fungi feed themselves quite ably, absorbing nutrients from organic materials. Algae and cyanobacteria are also adept at providing for their own nutritional needs by turning sunlight into energy through photosynthesis. Yet many thousands of years ago, some fungi merged with some algae (or cyanobacteria in some cases) to create a new kind of partnership called a lichen.

Some 20,000 different kinds of lichens live in such diverse habitats as the surfaces of rocks in arctic tundra and desert sands as well as the bark of trees in bayous and the sides of buildings.

Bacteria are very small. Yet despite their size they show a surprising degree of structural complexity. In this section we will look at the various compounds that make up a microbe and how these are put together. Learning the structure of a microbe helps in understanding how a microbe functions. To drive this point home, here are a few examples.

Disease causing bacteria (pathogens) have various structures that enhance their ability to cause illness. One important property is the ability to attach to the intended victim. pili, a proteinaceous surface structure on bacteria, are critical in this process.

Microbes are also capable of exchanging genetic information by mating. This process involves another type of surface structure, the F-pilus.

Bacteria will take steps to insure their survival. This can take the form of creating resting structures that allow the microbe to "sleep" during bad times. During abundant times, many microbes will store excess carbon, nitrogen, sulfur or phosphorous in inclusions in the cell.

Not only is structure important to understand functional relationships, it's also fascinating to observe what these little architects come up with.

There are some universal structures that all bacteria have. The basic building blocks of life, DNA, RNA, and protein, are common to all organisms not just microbes. Also, all microbes have a cell membrane. Much of what we know about these structures was obtained by studying bacteria, yet another reason to study them. Finally, most bacteria have a cell wall, but not all.

Whether measured by the number of organisms or by total mass, the vast majority of life on this planet is microscopic. These teaming multitudes profoundly influence the make-up and character of the environment we live in. Presently, we know very little about the microbes that live in the world around us because less than 2 % of them can be grown in the laboratory. Understanding which microbes are in each ecological niche and what they are doing there is critical for our understanding of the world.

Microbes are the major actors in the synthesis and degradation of all sorts of important molecules in environments. Cyanobacteria and algae in the oceans are responsible for the majority of photosynthesis on earth. They are the ultimate source of food for most ocean creatures (including whales) and replenish the world’s oxygen supply.

Cyanobacteria also use carbon dioxide to synthesize all of their biological molecules and thus remove it from the atmosphere. Since carbon dioxide is a major greenhouse gas, its removal by cyanobacteria affects the global carbon dioxide balance and may be an important mitigating factor in global warming. f, l, c, e. In all habitats, microorganisms make nutrients available for the future growth of other living things by degrading dead organisms. Microbes are also essential in treating the large volume of sewage and wastewater produced by metropolitan areas, recycling it into clean water that can be safely discharged into the environment. Less helpfully (from the view of most humans) termites contain microorganisms in their guts that assist in the digestion of wood, allowing the termites to extract nutrients from what would otherwise be indigestible. Understanding of these systems helps us to manage them responsibly and as we learn more we will become ever more effective stewards.

The vast majority of reported cases of foodborne illness occur as individual or sporadic cases. In most cases these originate, and occur, in the home. An outbreak occurs when two or more people suffer foodborne illness after consuming food from a contaminated batch.

Often, a combination of events contributes to an outbreak, for example, food might be left at room temperature for many hours, allowing bacteria to multiply which is compounded by inadequate cooking which results in a failure to kill the dangerously elevated bacterial levels.

Outbreaks are usually identified when those affected know each other. However, some are identified by public health staff from unexpected increases in laboratory results for certain strains of bacteria.

Energy is essential for our industrial society and microbes are important players in its production. A significant portion of natural gas comes from the past action of methanogens (methane-producing bacteria). Numerous bacteria are also capable of rapidly degrading oil in the presence of air and special precautions have to be taken during the drilling, transport and storage of oil to minimize their impact. In the future microbes may find utility in the direct production of energy. For example, many landfills and sewage treatment plants capture the methane produced by methanogens to power turbines that produce electricity. Excess grain, crop waste and animal waste can be used as nutrients for microbes that ferment this biomass into methanol or ethanol. These biofuels are presently added to gasoline and thus decreasing pollution. They may one-day power fuel cells in our cars, causing little pollution and having water as their only emission.

Microorganisms used in research have many useful properties. They will grow on simple, cheap medium and will often rise to large populations in a matter of 24 hours. It is easy to isolate their genomic material, manipulate it in the test tube and then place it back into the microbe. Due to their large populations it is possible to identify rare events and then with the use of powerful selective techniques isolate interesting bacterial cells and study them. These advantages have made it possible to rapidly test hypothesis in laboratory microorganisms that would have taken much longer in other organisms. Using microbes scientists have expanded our knowledge about life; here are a few examples.

In scientific research, microorganisms have been indispensable instruments for unlocking the secrets of life. The molecular basis of heredity and how this is expressed as proteins was described through work on microorganisms. Due to the similarity of life at the molecular level, this understanding has helped us to learn about all organisms, including ourselves.

A beer is any variety of alcoholic beverages produced by the fermentation of starchy material derived from grains or other plant sources. The production of beer and some other alcoholic beverages is often called brewing. Historically, beer was known to the Sumerians, Egyptians, and Mesopotamians, and dates back at least as far as 4,000 BC. Because the ingredients used to make beer differ from place to place, beer characteristics (type, taste, and colour) vary widely.

Typically, beers are made from water, malted barley, hops, and fermented by yeast. The addition of other flavourings or sources of sugar is not uncommon.

Because beer is composed mainly of water, the source of the water and its characteristics have an important effect on the character of the beer. Many beer styles were influenced or even determined by the characteristics of the water in the region.

Among malts, barley malt is the most often and widely used owing to its high enzyme content (which facilitates the breakdown of the starch into sugars) but other malted and unmalted grains are widely used, including wheat, rice, maize, oats, and rye.

Hops are a comparatively recent addition to beer (see History below). They contribute a bitterness that balances the sweetness of the malt and have a mild antibiotic effect that favours the activity of brewer's yeast over less desirable organisms. Enzymes in yeast, in a process called fermentation, metabolize the sugars extracted from the grains, producing many compounds including alcohol and carbon dioxide. Dozens of strains of natural or cultured yeasts are used by brewers, roughly sorted into three kinds: ale or top-fermenting, lager or bottom fermenting, and wild yeasts. Top-fermenting means that the yeast ferments in the top of the fermenting vessel. Conversely, bottom-fermenting means that the yeast ferments in the bottom of the fermenting vessel. The scientific name for ale yeast is Saccharomyces cerevisiae, an important model organism in molecular and cell biology; Saccharomyces carlsbergensis is the scientific name for lager yeast.

During the process of filtration (also called fining or clearing), some brewers add agents to beer that are not required to be published as ingredients. Since these finings may include animal extracts, vegans and others concerned with the use or consumption of animal products may wish to contact the brewer for specific details of the filtration process. Isinglass finings are a common animal-derived clarifying agent, extracted from fish. Alternatively, Irish moss is a commonly used plant-based clarifying agent.

One pint of beer typically contains about two to three units of alcohol, although alcohol content can vary significantly with style and brewer.

Almost any sugar or starch-containing food can naturally undergo fermentation, and so it is likely that beer-like beverages were independently invented in cultures throughout the world. In Mesopotamia, the oldest evidence of beer is on a 6000-year old Sumerian tablet which shows people drinking a beverage through reed straws from a communal bowl. Beer is also mentioned in the Epic of Gilgamesh, and a 3900-year old Sumerian poem honoring the brewing goddess Ninkasi contains the oldest surviving beer recipe, describing the production of beer from barley via bread. Beer became vital to all the grain-growing civilizations of classical antiquity, especially in Egypt and Mesopotamia.

Beer was important to early Romans, but during Republican times wine displaced beer as the preferred alcoholic beverage, and beer became considered a beverage fit only for barbarians. Tacitus wrote disparagingly of the beer brewed by the Germanic peoples of his day.

Most beers until relatively recent times were what we would now call ales. Lagers were discovered by accident in the sixteenth century when beer was stored in cool caverns for long periods; they have since largely outpaced ales in volume. (See below for the distinction.) The use of hops for bittering and preservation is a medieval addition. Hops were cultivated in France as early as the 800s. The oldest surviving written record of the use of hops in beer is in 1067 by Abbess Hildegard of Bingen: "If one intends to make beer from oats, it is prepared with hops." In 15th century England, an unhopped beer would have been known as an ale, while the use of hops would make it a beer. Hopped beer was imported to England (from the Netherlands) as early as 1400 in Winchester and hops were being planted on the island by 1428. The Brewers Company of London went so far as to state "no hops, herbs, or other like thing be put into any ale or liquore wherof ale shall be made — but only liquor (water), malt, and yeast." However, by the 16th century, "ale" had come to refer to any strong beer, and all ale and beer were hopped.

Methods of brewing changed very little from that time. In 1953, New Zealander Morton W Coutts developed the technique of continuous fermentation which was the first major change to brewing since the 16th century. Morton patented his process which revolutionized the industry by reducing a four-month long brewing process to less than 24 hours. His process is still used by many of the world’s major breweries today, including Guinness.

In 1516, the duchy of Bavaria adopted the Reinheitsgebot, perhaps the oldest food regulation still being used. The Reinheitsgebot ordered that the ingredients of beer be restricted to water, barley, and hops. The law soon spread throughout Germany, and has since been updated to reflect modern trends in beer brewing. To this day, the Reinheitsgebot is (controversially) considered a mark of purity in beers.

Some prokaryotes are capable of growing under unimaginably harsh conditions and define the extreme limits of where life can exist. Some species have been found growing at near100 °C in hot springs and well above that temperature near deep-sea ocean vents. Others make their living at near 0 °C in freshwater lakes that are buried under the ice of Antarctica. The ability of microbes to live under such extreme conditions is forcing scientists to rethink the requirements necessary to support life. Many now believe it is entirely possible that Jupiter's moon Europa may harbor living communities in waters deep below its icy crust. What may the rest of the universe hold?

Until recently, while we could study specific types of bacteria, we lacked a cohesive classification system, so that we could not readily predict the properties of one species based on the known properties of others. Visual appearance, which is the basis for classification of large organisms, simply does not work with many microbes because there are few distinguishing characteristics for comparison between species even under the microscope. However, analysis of their genetic material in the past 20 years has allowed such classification and spawned a revolution in our thinking about the evolution of bacteria and all other species. The emergence of a new system organizing life on Earth into three domains is attributable to this pioneering work with microorganisms.

The fruits of this basic research have been used by scientists to understand microbial activity and therefore to shape our modern world. Human proteins, especially hormones like insulin and human growth factor, are now produced in bacteria using genetic engineering. Our understanding of the immune system was developed using microbes as tools. Microorganisms also play a role in treating disease and keeping people healthy. Many of the drugs available to treat infectious disease originate from bacteria and fungi.

One last recent role of microbes in informing us about our world has been the tools they provide for molecular biology. Enzymes purified from bacterial strains are useful as tools to perform many types of analyses. Such analyses allow us to determine the complete genome sequence of almost any organism and manipulate that DNA is useful ways. We now know the entire sequence of the human genome, with the exception of regions of repetitive DNA, and this will hopefully lead to medical practices and treatments that improve health. We also know the entire genome sequence of many important pathogens and analysis of this data will eventually lead to an understanding of the function of critical enzymes in these microbes and the development of tailor-made drugs to stop them. The tools of molecular biology will also affect agriculture. The complete genome sequence of the plant Arabidopsis (a close relative of broccoli and cauliflower) is now known. This opens a new avenue to a better understanding of all plants and hopefully improvements in important crops.

Before we begin the adventure that we call learning microbiology (it can be thought of as an adventure!), a look at the history of microbiology will help you to understand the contributions of those who have come before. This perspective will hopefully give you an appreciation of their efforts and put the body of knowledge we will look at in the context of history. Keep in mind that microbiology is a relatively young science. It was only 130 years ago that it became possible to seriously study microorganisms in the laboratory with most of our understanding of microbes coming in the last 60 years.

The history of microbiology, like all human history, is not a catalog of linear progress, but is more of an interweaving of the careers of bright individuals and their insights. Each new discovery relied on previous ones and in turn spawned further inquiry. A web of interdependent concepts evolved over time through the work of scientists in many related disciplines and nations. Often the research of one individual impacted the efforts of another studying a completely different problem. Keep this in mind as you look at this history.

Below we present several journeys through this web, mentioning some individuals who were particularly important in the progression. This history reflects our view of important events of the past, but is by no means comprehensive. We will first look at the development of the techniques for handling microorganisms, since everything else in microbiology depends upon these procedures. Next, we will examine how these techniques helped to settle an old debate, the question of spontaneous generation. Then, we will look at the history of infectious disease. The science of microbiology had its most significant early impact on human health, discovering the cause of the major killers of the day, and then methods to treat them. As microbiology matured, scientists began to look at what non-pathogenic microbes were doing in the environment and we will look a bit at the history of general microbiology. Finally, the chapter will end with an examination of the events that lead to the understanding of life at the molecular level and the profound impact this has had on microbiology and on society in general. By examining a sequence of discoveries we hope to give you a sense of how science builds on itself. Notice especially how ideas in one path influence ideas in another.

Koch was convinced that microbes caused some diseases. However, to test this idea, he needed to isolate the causative agent. Almost all samples from diseased animals or any natural surface contained many different microbes and it was impossible to tell which one was the problem. A method was needed to separate these different bacteria. The most common method of isolation was to continually dilute a sample in liquid broth in hopes that at high enough dilution, only one type of microbe would be found. A problem with this method is that only the most populous microbe would be isolated, but that might not be one causing the disease.

There were other technical problems as well with such a liquid-based system, so a solid medium would provide distinct advantages. Koch has tried gelatin for these experiments with unsatisfactory results. Building on the work of Brefeld and Schroeter, Koch used potato slices as a solid medium and observed that a boiled potato left in the open air would develop tiny circular raised spots. c, g, l, c, c. Examination of these spots revealed they were made up of microorganisms and each spot had just one type of microbe in it. He realized that these colonies were pure cultures of bacteria and probably arose from a single species of microbe from the air that landed on the potato. By boiling a potato, slicing it with a hot knife and keeping it in a sterile container with a lid, Koch could keep the potato sterile. But if a sample from a disease animal was smeared across the potato, colonies arose, each being pure isolates from the animal. By then testing these isolates in animals, Koch was able to isolate the cause of anthrax, Bacillus anthracis.

Potatoes failed to support the growth of many microorganisms and Koch and his laboratory were constantly frustrated by the lack of a good solid medium. Walter Hesse joined Koch’s laboratory to do studies on air quality, showing a remarkable attention to detail and patience in his work. His wife, Angelina Fannie Hesse along with raising their three sons, also would assist her husband with his research in the laboratory. Walter was attempting to do his air quality experiments using medium containing gelatin as the solidifying agent. In the summertime temperatures would often rise above the melting point of gelatin. In addition, microbes would often grow in the cultures that were capable of degrading gelatin and in both cases this would cause liquefaction of the medium, ruining the experiments. One day the frustrated scientist asked Lina (as she was called) why her jellies and puddings stayed solid even in the hot summer temperatures. She told him about agar-agar, a heat resistant gelling agent that she had learned about while growing up in New York from a Dutch neighbor who had emigrated from Java. Development of the new agent by Angelina and Walter led to a resounding success. Few microbes are able to degrade agar and it melts at 100 °C yet remains molten at temperatures above 45°C. This allows the mixing of the agar with heat-sensitive nutrients and microbes. After solidification, it will not melt until a temperature of 100°C is again attained, allowing the easy cultivation of pathogens. It can also be stored for long periods of time allowing the cultivation of slow-growing microorganisms. Any type of broth can be mixed with agar and this gives great flexibility in the kinds of medium that can be made, thus many more types of microbes could be cultivated.

Koch laboratory also developed methods of pure culture maintenance and aseptic technique. Aseptic technique involves the manipulation of pure cultures in a manner that prevents their contamination by outside microorganisms and equally important prevents their spread into the environment. Remember that Koch was studying some of the most devastating microbial pathogens of the period and their release could potentially cause disease in the scientists working on them. These procedures were also absolutely critical because they allowed careful study of pure microorganisms, making it possible to identify the role of each microbe in a given situation.

Another problem in the cultivation of microbes was solved by Julius Petri while working in Koch’s laboratory. Solid medium was poured on glass plates and allowed to spread and harden. Once cooled it allowed a solid surface for streaking. However, creation of these plates required great care since exposure to the air (and the microbes in it) often lead to contamination. In addition, to prevent contamination of plates during incubation, a cumbersome bell jar was used. If one wanted to view samples, the plate had to be removed from the jar, further exposing it to unwanted microbes in the air. In 1887 Petri developed a shallow glass dishes, with one having a slightly larger diameter than the other. Medium is poured into the smaller dish and the larger one serves as a cover. This simple device solved all of the above problems and has taken on the name of its inventor, the petri plate.

These same techniques are essential in studying all microorganisms. Collectively the above techniques have been used to isolate and identify thousands of different microorganisms. As a testament to the significance of their achievement, these techniques are practiced with remarkably little change in every laboratory that works with microorganisms today. The table below lists some of the early advances that helped to develop the practice of microbiology.

Spontaneous generation is the hypothesis that some vital force contained in or given to organic matter can create living organisms from inanimate objects. Spontaneous generation was a widely held belief throughout the middle ages and into the latter half of the 19th century. In fact, some people still believe in it today. The idea is attractive because it meshed nicely with the prevailing religious views of how God created the universe. There was a strong bias to legitimize the idea because this vital force was considered a strong proof of God’s presence in the world. Many recipes and experiments were offered in proof. To create mice, a recipe called for dirty underwear and wheat grain be mixed in a bucket and left open outside. In 21 days or less, you would have mice. The real cause may seem obvious from a modern perspective, but to the proponents of this idea, the mice spontaneously arose from the wheat kernels.

Another often-used example was the generation of maggots from meat that was left in the open. The failing here was revealed by Francesco Redi in 1668 with a classic experiment. Redi suspected that flies landing on the meat laid eggs that eventually grew into maggots. To test this idea he used three pieces of meat. One piece of meat was placed under a piece of paper. The flies could not lay eggs onto the meat and no maggots developed. The second piece was left in the open air, resulting in maggots. In the final test a third piece of meat was overlaid with cheesecloth. The flies were able to lay the eggs into the cheesecloth and when this was removed no maggots developed. However, if the cheesecloth containing the eggs was placed on a fresh piece of meat, maggots developed, showing it was the eggs that "caused" flies and not spontaneous generation. This helped to end the debate about spontaneous generation for large organisms. However, spontaneous generation was so seductive a concept that even Redi believed it was possible in other circumstances.

The concept and the debate were revived in 1745 by the experiments of John Needham. It was known at the time that heat was lethal to living organisms. Needham theorized that if he took chicken broth and heated it, all living things in it would die. After heating some broth, he let a flask cool and sit at a constant temperature. The development of a thick turbid solution of microorganisms in the flask was strong proof to Needham of the existence of spontaneous generation. Lazzaro Spallanzani later repeated the experiments of Needham, but removed air from the flask, suspecting that the air was providing a source of contamination. No growth occurred in Spallanzani’s flasks and he took this as evidence that Needham was wrong. Proponents of spontaneous generation discounted the experiment by asserting that air was required for the vital force to work.

It was long suspected that living things were the agents of disease. In volume 6 of his epic poem De Rerum NaturaI (On the Nature of the Universe) written sometime around 50 B.C., Titus Lucretius Carus speculates about invisible atoms causing disease. This was only one idea among many and some thought that an imbalance in humors caused illness, while others felt that supernatural forces were at work. The prevailing theory held by most doctors of the 19th century was that chemical toxins were carried from an ill patient to others, causing them to contract the same malady. The bacteria that were known to be present were seen as a symptom of the disease and not its cause. h, c, g, a, j, d, l, a, i. Ignaz Semmelweis, a Hungarian physician working in Vienna, made the first breakthrough in the true nature of disease. He realized that asepsis in obstetrical wards could prevent the transmission of childbirth fever from patient to patient and instigated a policy for all attending physicians to wash their hands with with chloride of lime (a mixture of calcium chloride hypochlorite, CaCl(OCl); calcium hypochlorite, Ca(OCl)2; and calcium chloride, CaCl2) between patients. This innovation dropped the mortality rate or mortality from 18% to 2.4%. Ignaz became a vigorous proponent of these ideas, but the Hungarian doctor's efforts were opposed by many who could not accept that physicians themselves could be responsible for spreading bacterial infection. Ridicule of his idea caused him to move from Vienna to Pest, Hungary and ultimately played a role in a nervous breakdown. Ironically Semmelweis died from an infection that he contracted during a surgery he performed, while recovering from his nervous breakdown. Before his death he published his ideas in a paper The Cause, Concept, and Prophylaxis of Childbed Fever in1861. The work was then ignored for 17 years, which raises an interesting point about the culture of science. Radical ideas, even those that are correct and can save lives, are sometimes ignored. It takes time to overcome the dogma of the day. The personalities involved and the negative light it might throw on past practice play a large role in the rate of acceptance of a new idea. His book was also very confusing dues to its poor writing and this also contributed to the obscurity of his ideas.

Koch's postulates

1. The specific organism should be shown to be present in all cases of animals suffering from a specific disease, but should not be found in healthy animals.

2. The specific microorganism should be isolated from the diseased animal and grown in pure culture on artificial laboratory media.

3. This freshly isolated microorganism, when inoculated into a healthy nonimmune laboratory animal, should cause the same disease seen in the original animal.

4. The microorganism should be reisolated in pure culture from the experimental infection.

This is his most famous contribution to science and it is a testament to the utility of these postulates that they are stilled used today to discover the cause of new emerging diseases. Koch went on to apply these principles in the study of many other diseases including tuberculosis, cholera and sleeping sickness. It should be pointed out that Koch’s postulates cannot be applied to all diseases. For example if a disease-causing microbe has humans as its sole host and has a significant possibility of causing death, it would be unethical to apply this microbe to test humans as dictated by postulate 3. Also, it is not always possible to obtain a disease-causing microbe in pure culture.

Koch developed the tools for obtaining pure cultures to attack the problem of disease. Advances in science often come from innovations in the available technology. Robert Koch was an important microbiologist because his pioneering work in the isolation and characterization of bacterial diseases helped to identify the causes of many of the maladies plaguing humanity. Further work by other scientists then began the long road to conquering them.

Smallpox was a feared disease throughout human history and justifiably so. It was highly contagious and almost everyone eventually became infected. Mortality rates were as high as 25% in adults and closer to 40% in children. Those who did survive often had scarring due to the blister-like pustules that form on the skin, but they obtained life-long immunity to the disease. As far back at the 11th century in India and China it was realized that liquid from the pustules of a smallpox victim, when scratched on the skin of a healthy patient, would most often cause mild disease. This intentional infection, termed variolation, would also give life-long protection against the virus. Lady Mary Wortley Montgue, wife of ambassador to the Ottoman Empire, introduced variolation to England in 1721 and it became a popular practice throughout Europe. Washington even began variolating the Continental Army in 1776.

Variolation had some deleterious side effects. Serious skin lesions inevitably resulted at the site of inoculation, often accompanied by a generalized rash or even a full case of smallpox. The fatality rate from variolation was 1 to 2 %. Today we would find this level of fatality to be unacceptable, but this risk still represented a significant advance. In the 1796, Edward Jenner, an English country physician, went in search of a more predictable and safer method of protection against the disease. He noticed that milkmaids rarely contracted smallpox. Further investigation revealed they often contracted cowpox from their charges. In a perceptive leap of faith, Jenner hypothesized that cowpox was related to smallpox and contraction of the former would protect against the latter. In a classic experiment (and one that would land you in jail today), Jenner inoculated a young patient with cowpox and later challenged him with smallpox. The boy did not become ill and Jenner was responsible for the creation of a safer method of protection against small pox. It is important to stress that the nature of these diseases and their viruses would not be known for over 100 years. Jenner was ahead of his time.

Beginning around 1876 Pasteur's studies on chicken cholera led to the development of vaccines to fight the disease. Cholera was a serious problem since it was able to spread through a barnyard and wipe out a flock in as little as 3 days. It was transmitted by contaminated food or animal excrement. Pasteur had identified the cholera bacillus and was growing it in pure culture. When injected with it, a chicken invariably died within 48 hours.

Then, as often happens in scientific research, luck intervened. During the heat of the summer, Pasteur returned to Paris and left the cholera cultures used for infection stored on the shelves of his laboratory in Arbois, France. Upon returning, something had happened to the cultures, they no longer caused disease when tested in chickens. With some impatience for the time they were wasting, his group set to work making new cultures of the bacillus and tested these batches on both new birds and also those previously inoculated with the ineffective strain. To their amazement the previously injected birds were unaffected by the fresh bacillus culture, while the new birds all died. Pasteur immediately realized that this was similar to the studies of Jenner. Pasteur then developed a method for creating cultures that would confer immunity, but not cause disease. In honor of Jenner's accomplishments Pasteur coined the term vaccination (vacca = cow in Latin) for the process of immunization against disease. In this and several of Pasteur's other discoveries, luck played a part, but it was only helpful because he tenaciously pursued "odd" results and had the insight to arrive at important conclusions. In Pasteur's famous words, " In the field of observation, chance favors only the prepared

We now pick up another thread though the web of change that began with the work of Paul Ehrlich. By 1885 it was becoming clear that the causative agents of many illnesses were microorganisms. As scientists manipulated these microbes in the lab they found that certain dyes and other compounds were able to inhibit their growth. This inspired Ehrlich to propose that chemicals may exist that will kill the microbe, but not the patient. The hope was that by having a patient take these chemicals they could cure the illness. One of the diseases he hoped to cure was syphilis, which had reached epidemic proportions in Europe. Little did he realize it would be a 17-year odyssey before he would develop salvarsan, the first effective chemotherapeutic agent. Salvarsan was the 606th chemical he tried and is an arsenic compound that effectively kills Treponema pallidum, the causative agent of syphilis. However, the treatment had many problems, causing long lasting health complications for those individuals who used it.

In addition, despite Treponema being quite sensitive to salvarsan, the physician had to administer it intravenously for optimum effectiveness. This type of injection was a recent development and many doctors were leery of trying the procedure. In London a young physician by the name of Alexander Fleming, then in the Army Medical Corps, was one of the few that was willing to treat patients, even getting the nickname private 606 from his burgeoning practice. His work validated the effectiveness of salvarsan against syphilis and convinced others to try the treatment. a, i, a, l. Fleming was a physician by training, but spent most of his time studying bacteria, and his success with salvarsan motivated him to search for other antibacterial agents. His first discovery was lysozyme, an enzyme produced by many organisms, including humans, that lysed some bacteria. This enzyme is not useful as a therapeutic agent because it is difficult to administer as a drug, but Fleming did develop titration methods and assays that would become very useful. In September of 1928, before leaving on a summer holiday, Fleming streaked some plates of Staphylococcus aureus and left them to incubate until his return.

In an improbable set of circumstances, the beginning of the holiday was cold, allowing some contaminating mold spores (that had blown in from a nearby window) to grow up on some of the plates. The temperature then increased encouraging the growth of the Staphyloccoccus. Many experimenters when confronted with a contaminated plate will look for the trash bin, but Fleming instead spent some time examining it. The fungus had a zone of clearing around it where the Staphylococcus colonies would not grow, suggesting the fungus was producing an antibacterial compound that had diffused into the medium. Intrigued, he cultured the fungus, a Penicillium mold, and eventually isolated a soluble extract that could kill bacteria and treat localized infection. He called the new compound penicillin after the mold it came from. Due to the technology available, it was very difficult to prepare a solution that could be used throughout the body without causing problems.

World War II added a greater urgency to the search for compounds that could fight infectious disease. Wounded soldiers, if they survived the initial injury, would often develop life-threatening infections and there were no effective drugs to combat them. In 1939 Howard Florey and Ernst Chain began a systematic study of antimicrobial compounds in hopes of developing treatments for these soldiers and ran across Fleming's report written 9 years earlier. They now were able to purify the compound completely and describe its high potency against microbes. The availability of penicillin during World War II saved countless lives. The rediscovery of penicillin touched off a search for other microbes producing substances that could kill or inhibit microbes, leading to the discovery of many more antimicrobials. With Florey and Chain, Fleming was awarded the Noble prize in Medicine and Physiology in 1945.

Martinus Beijerinck was originally trained as a botonist and began his work studying the microorganisms that were present in and around plants. He soon began experiments with microbes in the soil. His greatest contribution was the development of enrichment media. Previously, microbes were cultivated on medium consisting of potatoes or extracts of left-over animal renderings. Such media would support the growth of many different bacteria, with chance and population density dictating what became dominant in the culture. In many cases this was exactly what was desired, but in other cases it was of interest to find bacteria capable of performing certain chemical conversions.

Beijerinck discovered that by adding or removing certain compounds from the medium or incubating under different conditions, it was possible to favor the growth of certain microbes and prevent the growth of others. An example is Beijerinck's work with nitrogen-fixing bacteria, which are important in agriculture and the global cycling of nitrogen. They are capable of taking nitrogen gas (N2) from the air and reducing it to ammonia (NH3). This is an important property since reduced nitrogen compounds such as ammonia are the only compounds most organisms, including agriculturally important plants, are able to use as a nitrogen source. j, g, j, h Beijerinck wanted to isolate an organism capable of fixing nitrogen in the presence of air, because all previous isolates fixed nitrogen only under anaerobic conditions. By making medium that did not contain a source of fixed nitrogen and then incubating in the presence of air (containing N2), he demanded that any microbe growing in the medium had to be able to derive its nitrogen by performing aerobic nitrogen fixation. By this method he succeeded in isolating a new microbe (Azotobacter chroococum) with these capabilities. Similar selective culture techniques (as he liked to call them) enabled his laboratory to isolate sulfur-reducing and sulfur-oxidizing bacteria, Lactobacillus species, green algae and many other microbes.

Sergei Winogradsky was also interested in soil bacteria, especially those involved in the cycling of nitrogen and sulfur compounds. He was one of the first to isolate microorganisms responsible for the conversion of these elements in the soil, obtaining pure cultures of bacteria capable of the conversion of ammonia to nitrate by microorganisms in the soil. Winogradsky also studied the consumption of hydrogen sulfide gas by sulfur-oxidizing (Check this!!!!) bacteria directly in their natural habitat.

When working with Beggiatoa (one of these sulfur-oxidizing bacteria) he discovered it was obtaining its cell carbon from carbon dioxide. He used the term autotrophy to describe this property, a radical idea at the time. This led Winogradsky to propose the concept of chemolithotrophy - bacteria capable of growth using purely inorganic sources of carbon and energy. He realized the impact these processes have on our environment long before others accepted the idea.

As a result of the work of Winogradsky and Beijerinck there was great enthusiasm for identifying and classifying the bacteria inhabiting our natural world. For the first part of the 20th century many scientists isolated microbes and made proposals for their organization into genera and species. In 1909 Sigurd Orla-Jensen suggested a classification scheme based on the functions and abilities of the bacteria, such as growth on certain compounds or the production of specific by-products. The Society of American Bacteriologists, later to become the American Society of Microbiology, applied this technique to prepare a report on the classification of bacteria. This eventually evolved into Bergey's Manual of Determinative Bacteriology in 1923 and subsequent editions have become authoritative reference works. There was great optimism that as more bacteria were brought into pure culture a clear organization, based on the evolutionary history of microbes, would emerge from studies of their physiology. However, y the 1940's it became clear that bacteria were unwilling to go along with this idea because classification based on one set of properties was often inconsistent with classification using a different set of properties. Scientists threw up their hands and gave up the idea of finding a system of classification based on growth and morphological characteristics.

Several decades passed during which bacteria became tools for understanding life, but their place in evolution was of little interest. Then in 1961 McCarthy and Bolton developed methods for the comparison of genetic material between species and Pauling and Zuckerkandl formalized the idea of using the makeup of biological molecules, their sequence, as a way to determine phylogenetic relationships. Later, Carl Woese began to use this insight, choosing the sequence of the 16S rRNA from the ribosomes of bacteria as basis for evolutionary comparisons. This analysis was a watershed event for the evolutionary classification of bacteria and in 1977 Woese used this technique to assert that known bacteria should be divided into two separate domains that we now call Bacteria and Archaea, radically changing our view of the microbial world. As you will read in the following chapter (Microbial Structure), Lynn Margulis proposed the remarkable notion that some bacteria fused with archaea to crate eukaryotes. This hypothesis, termed endosymbiosis, has been strongly supported by a range of subsequent biochemical data. In subsequent years, determination of 16S rRNA sequences, and then complete genomes, of microorganisms has enabled the natural classification of microorganisms. A review by Olsen, Woese and Overbeek provided a catalyzing summary of the tree of life for microbes, including eukaryotic microorganisms. This reorganization has reshaped our view of the tree of life for all organisms and helped to reassert the important role of microbes in shaping the past and affecting the future of the earth. A renewed interest in environmental microbiology, using new molecular tools based often on 16S rRNA, is revealing much more about the microbes present in our world and their influence on it.

In the early part of the 20th century techniques were developed to examine the inner workings of the cell and much of the work was performed in bacteria due to their experimental accessibility. Before this period, the method of turning genetic information into the proteins that carry out cellular processes was completely unknown. Indeed, the very nature of genetic material was being hotly debated when in 1928 Fred Griffith discovered transformation in bacteria.

Griffith knew that when Streptococcus pneumoniae are injected into mice, they cause rapid deterioration and death. Griffith isolated strains of the bacteria that were no longer capable of producing an outer slime layer and appeared rough when grown on solid medium in contrast to the smooth colonies of the original isolate. When these rough strains were injected into mice, no illness resulted. Similarly, when a smooth microbe was heat-killed and then injected, the mice showed no signs of infection. A surprise was waiting for Griffith when he mixed the dead cells of a smooth isolate with live cells of a rough isolate and then injected this into mice; they died. When bacteria were isolated from these dead mice, they were all smooth. Griffith hypothesized that the ability to create the slime layer was passed from the dead smooth cells to the viable rough cells, making them pathogenic again. The process became known as transformation. He was ridiculed, as most scientists believed his preparation were contaminated with viable smooth cells. It was not until 1944 that his student Oswald Avery and coworkers repeated the experiments of Griffith, reproducing his results and discovering that DNA was the material from the dead smooth cells that transformed the rough mutant. This was strong proof that the hereditary material in cells was DNA, though the actual structure of DNA was not to be discovered for years. Further experiments in the 1940's clearly established this observation with Joshua Lederberg discovering two more ways that DNA could be transferred between bacterial cells, conjugation and transduction.

In 1943 Beadle and Tatum reported experiments with the fungus Neuropsora crassa that eventually established the idea that each gene in the DNA typically codes one protein (the “one gene-one enzyme” hypothesis). About 10 years later, James Watson and Francis Crick wrote their landmark letter to the journal Nature describing the structure of DNA and making predictions about how it was replicated. It was now clear that DNA stored the information for proteins and that proteins performed the many functions of the cell. The important question now became, how does one convert the information in DNA into protein. A major contribution in understanding this puzzle was made by Paul Zemecnik and his lab who developed cell-free systems, first with rat liver and later using the bacterium E. coli. This allowed the study of translation in the test tube and He and other scientists used these systems to discover the important molecules involved in the process. An intense effort to describe these molecular events then ensued. The first light came when Crick, Sydney Brenner and colleagues proposed the existence of transfer RNA (tRNA), a molecule that helps to create amino acid polymers based on nucleic acid sequence. Another critical player in the processing of information was revealed when Brenner, Francois Jacob and Matthew Meselson discovered that translation of genetic material into protein takes place on the ribosome and that the molecule being translated at the ribosome is RNA, not DNA. The next mystery was solved by Marshal Niremberg and J. H. Matthaei when they developed methods to decipher the genetic code that dictates the correspondence of nucleic acids to amino acids. After the stunningly productive decade of the 1960's, the nature of the framework for the conversion of the genetic information into proteins was now understood and the basic mechanism has since been shown to be conserved across all biology. Note that almost all of this work occurred in microorganisms.

In 2001, the Center for Science in the Public Interest (CSPI) petitioned the United States Department of Agriculture to require meat packers to remove spinal cords before processing cattle carcasses for human consumption, a measure designed to lessen the risk of infection by variant Creutzfeldt-Jakob disease. The petition was supported by the American Public Health Association, the Consumer Federation of America, the Government Accountability Project, the National Consumers League, and Safe Tables Our Priority. This was opposed by the National Cattlemen's Beef Association, the National Renderers Association, the National Meat Association, the Pork Producers Council, sheep raisers, milk producers, the Turkey Federation, and eight other organizations from the animal-derived food industry. This was part of a larger controversy regarding the United States' violation of World Health Organization proscriptions to lessen the risk of infection by variant Creutzfeldt-Jakob disease

The discipline of bacteriology evolved from the need of physicians to test and apply the germ theory of disease and from economic concerns relating to the spoilage of foods and wine. The initial advances in pathogenic bacteriology were derived from the identification and characterization of bacteria associated with specific diseases. During this period, great emphasis was placed on applying Koch's postulates to test proposed cause-and-effect relationships between bacteria and specific diseases. Today, most bacterial diseases of humans and their etiologic agents have been identified, although important variants continue to evolve and sometimes emerge, e.g., Legionnaire's Disease, tuberculosis and toxic shock syndrome.

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