Observing bacteria, fungi and protozoa using the light microscope.

Bacteria have been around on this planet for something in the region of 3.5 billion years in some shape or form. When the earth formed from accreted planetesimals that were formed from the solar nebulae some 4.5 billion years ago it was a very hot ball of molten rock and metal. Yet within a few hundreds of millions of years the earth had cooled sufficiently to allow liquid water to form oceans. The oceans and land masses were nothing like the oceans and continents that we know today. Other star systems have been observed with what is known as a protoplanetary disc surrounding them. Beta Pictoris seems to be such a candidate at a distance of 600 million million Km and with a disc some 900 AU across. Beta Pictoris is about 63 LY distance with a surface temperature of 8000 K and a solar radius of 1.9. This disc of material may provide the ingredients for a future habitable world

It seems the earth was the only planet in our solar system to sustain life of any sort because the conditions were just right at that time. Plate tectonics, tilt of the earth, distance from the sun, stability and type of star, the right chemical balance, amount of water all of these ingredients played a major factor in allowing inorganic material to become organic. Life or the traces of life have been recovered from the rocks by scientists using techniques that would have astounded Charles Darwin and his contemporaries. Darwin always had the problem of explaining how complex metazoans suddenly appeared in the fossil record. Unbeknown to Darwin hidden from prying eyes were the fossil remains of single celled organisms just waiting for the time when human ingenuity could find a way of extracting this evidence and having the necessary skills to be able to read the story of the rocks. The evidence for ancient life is now overwhelming and most scientists now accept that the planet and the organisms that are on it are of a very ancient lineage.

The age of the Earth is about 4.5 billion years old and for 3 billion years single celled organisms have ruled the roost. Through their unique adaptability to different biomes, they have over the aeons transformed the Earth along with plate tectonics and other natural phenomena, into a place where large multi celled creatures can endure for millions of years. The suns energy output must have been fairly constant over this period in order for life to have survived for so long. How many other stars have this quality and also the amount of metallic ingredients to seed its system with rocky planets? The planet would need to be at a certain distance from its star and also be protected from the solar radiation that would continually rain down upon it. In order to protect any life from the deadly solar radiation the planet would need a fluid outer metallic core of the right size in order to have a magnetic field surrounding the planet. The planet would also need the right amount of water and also dry land. Would life start in the shallow seas or deep down in the ocean? Would it need a moon and would the planet fare better if it were tipped over similar to the Earths 23o in order to have variable seasons.
The most important question that we could ask is whether animal life would eventually emerge and with that possible intelligence. Looking at the development of large animals like our selves and all the other multi celled organisms that have at some time inhabited the Earth, it seemed very difficult for metazoans to get established. Would this apply to all systems with habitable planets? If an alien spacecraft were to have surveyed this planet 3-4 million years ago would they have guessed that the little ape walking on the African plains would one day also travel into space. Lady luck is a major player in the game of evolution by natural selection. Humans are no doubt unique on the Earth but so are lions, chimps, birds and fish, whether we like it or not we are just another player on this ball of rock.. You do not need intelligence to survive but it does make for a much more rewarding experience while you are here. The twists and turns that went about on the Earth are unimaginable and if the dice were rolled again would we get the same results, I think not.

At the moment there is much interest in the search for life and also possible life bearing planets. The British science group led by Professor Alan Wells (Lander Operations Control Centre, University of Leicester) are at this moment trying to make contact with the Beagle spacecraft. It would seem however that Beagle as gone the way of many other Mars spacecraft and simply crashed or landed in an awkward place unable to send any transmission to the mother ship orbiting above. The chances of finding any extant life on Mars must be zero, but maybe the traces of fossil life (microbes) might just be there waiting to be prized free by some enterprising group of scientists, you just never know. I personally do not believe that there has been life anywhere else within our solar system. I also believe that metazoans and intelligent life are almost non existent elsewhere in our part of the galaxy and to extrapolate beyond that and to other galaxies is pointless. I do believe however that the likely hood of single celled organisms on other worlds is quite high and it would be very interesting to try and discover if they have a similar biochemistry to life on the Earth. Scientists now have the technology to discover other planets orbiting around other stars. Most of these planets are Jupiter sized and also orbiting much closer to their parent star than Jupiter is to our sun. This would definitely negate the possibility of earth like planets in this system, and would also throw doubts on our solar system being the standard model. In the near future, 10-20 years time it may be possible to detect earth like planets orbiting around other star systems. Scientists may also be able to detect oxygen, methane and ozone within these planetary atmospheres using spectroscopy. The possibility of humans travelling to these star systems has to be about zero so it looks like the only way to communicate over such large distances is by radio. In truth I think that would be the easy bit, the chances of finding something similar to us in the same time period must be extremely slim. Would we be able to recognise different life forms and their communications? It also helps to bear in mind that these creatures may not not be vaguely interested in identifying themselves. And there is the other more scary reason that we might just be the only sentient beings in the galaxy and possibly the universe.

Freshwater microbes X40 phase contrast.

Because of their size and almost transparency, bacteria are very difficult to observe with an ordinary light microscope and therefore need to be either stained with a dye, or some form of optical enhancement needs to be employed. Staining bacteria with the use of dyes as been around for over a 150 years and has proved to be an excellent tool for identification, especially for medical diagnostic use. The gram stain, which was developed by a bacteriologist called Hans Gram over a century ago is still the main stain used in microbiology. It works by staining the cell cytoplasm either purple (gram positive) or pink (gram negative).
Another way to show up the bacteria is to use optical contrast techniques such has phase contrast, darkfield or differential interference contrast. These techniques allow the observer to view the micro organism in much clearer detail than would be possible with ordinary brightfield objectives. The only problem with phase contrast is that a halo of bright light surrounds objects and therefore can be confusing to the inexperienced observer when trying to measure or make out very fine detail within the cell. Because it does show a great deal of cell differentiation it is worth the amount of money and anyone who finds themselves drawn towards the study of bacteria or even protozoa and algae would do well to invest in such a piece of equipment.
There are two types of single cells on earth today; prokaryotes, which include bacteria (cyanophytes) and archaea and the eukaryotes, which include the fungi, animals, algae, protozoa and the larger plants. The bacteria are prokaryotes which do not have a membrane surrounded nucleus; their DNA is not surrounded by a membrane but left loose and irregular and does not have Histones (proteins) associated with the DNA. There are no membrane bound organelles and their cell wall is made up from peptidoglycan which is a complex polysaccharide. Bacteria divide by splitting into two parts, each daughter cell identical to the other, this is called binary fission. Bacteria are also of a much smaller size than eukaryotic cells. Prokaryotic cells ruled the earth for something like 3 billion years until they gave rise to the much larger and more complex cells of the eukaryota.

Micro organisms have, over the billions of years transformed the earth into a place that has teemed with life from our humble single celled bacterium to mighty giants like Tyrannosaurus rex and the blue whale. They (bacteria) along with plate tectonics transformed the atmosphere from a poisonous canopy of CO2 to one that contains nitrogen and oxygen. Over the millennia many creatures have come and gone but the bacteria have been here from day one and will probably be the only organisms around to take the final bow.
The bacteria that humans are mostly concerned with for obvious reasons are those that cause disease and also the ones that attack our crops. Bacteria are part of us, in fact there are more bacterial cells within and on the human body than there are human cells. So why don't these enormous number of bacterial cells destroy us from the word go. Many of the bacteria found living within us are of paramount importance to our well being, producing vitamins and keeping other more pathogenic bacteria at bay. It is only when microbes enter those parts of the human body that they do not belong that problems with disease begin to appear.
The human mouth is home to something in the region of 300 species of bacteria, with a few species of fungi and protozoa thrown in for good luck. When first born humans are completely bacteria free. They acquire there flora and fauna from their mother at birth and any one else who happens to breathe on or put a loving arm around the baby. Very shortly the mouth will be teeming with microbes, possibly hundreds of millions. This does not mean that the mouth is a nasty place, quite the contrary, especially if you happen to be a bacterium. Let's think about this for a second. Where else could you set up home and have a nice comfortable liquid environment with more food than you can shake a stick at. All the warmth and protection you might need for many years while you expand your population.

X63 Phase contrast.

The above photograph is part of an original image which was taken with a phase contrast objective. The bacteria have adhered to the cell surface of an epithelium cell and appear to be dividing. These cells can be found by scraping the inside of the mouth with a clean finger and making a wet mount. Plenty of bacteria may be found on some, but not all of the epithelium cells that will be scattered through out the smear.
A good way of actually viewing these various micro organisms is to do a little experiment that a man called Antony Van Leeuwenhoeck did back in the 1600s. Refrain from cleaning your teeth for just a day and eat chocolate and any other sugary foodstuffs (good excuse). This single day of not cleaning your teeth should do no lasting damage but may feel a bit uncomfortable. Leeuwenhoeck took scrapings from his own mouth and also from other peoples mouths and stumbled on a teeming world of bacteria or animalcules has he preferred to call them. He also drew as many examples has he could and sent is report to the Royal Institution here in London. Well those same species that he drew are still here with us and can be easily found by carrying out Leeuwenhoecks simple experiment. Using a toothpick gently remove some of the plaque from between the teeth and smear it across the glass slide, add some water and lower your coverslip over the smear. This type of mount is called a wet mount and can be also used for pond life. If you are using phase contrast then you should begin to observe very many different types of bacteria, from spirochetes, long rods, short rods and cocci. Many will be seen to whiz around while others just lazily wiggle along, there are also many species that are non motile. While you are observing take a look at the two fundamental types of cell. Compare the eukaryotic cell (cheek cell) to that of a prokaryotic cell (bacterium) the size difference is enormous.( Prokaryotic cells 0.2- 2µm in size ) ( Eukaryotic cells 10- 500µm in size )

These types of bacteria are very common in freshwater habitats and are quite large and readily observable with the aid of phase contrast. A micro organism similar to this specimen, Treponema pallidum, is responsible for causing the debilitating disease syphilis in humans. Another spirochete that can cause serious illness and even death is Weil's disease, again caused by a spirochete that is shed into freshwater sites via rat's urine. When searching for microbes around quite still waters always wear gloves that are water proof and never touch eyes, ears or mouth with your hand. Spirochetes similar to the one shown above occur in their millions within the human mouth.
Another interesting microbe that can be found living quietly within the oral cavity is a protozoan called amoeba gingivitis. This microbe is an amoeba and can be found living within the cavities that sometimes occur within the mouth.

X40 phase contrast.

It is not sure whether these small amoebas are pathogenic or whether they help by cleaning up the bacteria and other waste products found within the mouth.
Another type of organism that can invade the body especially when the immune system is compromised by AIDS, or chemo therapy for instance is a fungus called Aspergillus. The most important nosocomial infection is Aspergillus spp, which is usually inhaled as spores that may settle within the lungs causing pneumonia. This filamentous fungus will eventually spread to other parts of the body via the blood supply system. There are over 150 species of this genus but only about twenty are opportunistic agents of disease in humans.
Photograph (A) shows a colony of Aspergillus in a section of human lung, while photo (B) shows an unidentified species of fungi which had been grown on an agar plate. The white line in B represents 1cm.

The fungi that cause intense itching and soreness around and between the toes that generally goes by the name of athletes foot is an opportunistic pathogen. Many people suffer from this fungal infection and once caught can be almost impossible to eradicate completely.

Some bacteria (bacillus & clostridium) have ways of surviving inhospitable environments that would otherwise destroy them.Most bacteria will quickly die in temperatures above 70-80 C but the hay bacillus endospore can resist temperatures at 100C and still survive. Forming resistant spores is one good way to survive until conditions improve. Spore formation can also be a diagnostic tool to identify certain spore forming bacteria. The bacterium copies its own genetic material and forms a tough coating around the DNA while the rest of the cell disintegrates. The cell can sit out the adverse conditions with no metabolic activity, sometimes for thousands of years surviving extremes of heat and cold. Some scientists believe that it is possible for bacteria in the shape of spores to travel from planet to planet embedded within rock fragments that have been blown clear of the gravitational pull of the planet. This scenario could happen when a large meteor or comet collides with a planetary body throwing pieces of the crust clear. Whether any microbes could withstand such an impact and the possibly millions of years in deep space is a matter of debate. Also the microbes would also have to survive the re-entry into the new world's atmosphere, and this would be dependent on the size of the piece of rock and thickness of the atmosphere.

To show how resilient some bacteria can be a certain species even went to the moon and back and survived the vacuum and extremes of temperature that are only found in the vacuum of space. There are microbes that survive boiling temperatures and enormous pressures in the deep oceans of the world living without sunlight for all their lives. The early conditions on the earth were probably very hot by today's standards and also no free oxygen in the atmosphere; meant that these microbes were truly alien to most of the bacteria that we know of today. The surface of the planet would have been scorched with ultra violet radiation from a star that pumped out much less light and heat than it does today. What type of bacteria were around then is probably lost forever in deep time, but they must have been very tough organisms indeed. It is speculated that the black smokers that scientists discovered in the 1970s may have been the ideal place for the first life forms to have originated, being protected from harmful UV light and also possible damage from large bodies crashing into the earth. Again all this is speculation.

Some species of bacteria have what are called flagella which provide them with a means of moving from one part of the environment to another. There are four different arrangements of flagella which can go from a single flagellum to the cell being completely covered with flagella. The flagella are extremely complex and consist of three major regions: - basal body, hook and filament. It is only in the later part of the last century that scientists began to get some ideas as to how this mechanism worked. Flagella are very difficult to observe under the light microscope and it is usual for microbiologist to use special stains which adhere to the flagella increasing their diameter. A good species for showing peritrichous flagella (covering the entire cell) is Proteus vulgaris. Some bacteria can protect themselves by secreting large amounts of mucilage around themselves and thereby preventing desiccation or even attack from predators. The two different species below were found in fresh water.

The bacterium below is probably Beggiatoa alba, which grows in most freshwater habitats between the interface of aerobic and anaerobic layers. It uses hydrogen sulphide (H2S) as an energy source and is therefore a chemolithoautotroph. These bacteria usually give their identity away by the large white mats that they produce when reproducing. The granules that can be just seen within the cell body are Sulphur. This microbe was important in the discovery of the autotrophic metabolism.

These two photographs show different species of sulphur bacteria.

The photographs below are of a colony that consists of millions of individual bacteria. The bacteria were grown on an agar plate specially prepared under sterile conditions and then infected with bacteria either from the hand,mouth or nose. After just a few days at room temperature spots begin to appear on the red agar plate and very quickly grew in size to reach a few centimetres across. The photograph on the left was taken with a coolpix 4500 using a Wild stereo microscope with a special adaptor in place to accept the camera. The second photograph (also taken with the Nikon coolpix 4500) is a high power phase contrast photo and shows only the shape of the microbes, but very little else.

When growing bacteria on agar plates, some amazing shapes and colours occur and can be very beautiful to look at. But it must be remembered that these are potential pathogens and should be treated with the greatest of respect. Once finished with, the plates should be incinerated and completely destroyed.

This is another colony of bacteria grown on an agar plate.

This photograph was taken from a yogurt culture and shows a streptococcus bacterium.

All the photographs were taken by Stephen Durr on various types pf microscopes. Zeiss photomic 111 and Leica dialux and Orthoplan microscopes fitted with brightfield, phase contrast and differential interference contrast objectives.

Some books on microbiology that you may find useful.

A field guide to Bacteria. This book was written for the serious amateur and student of microbiology. Betsey Dexter Dyer is a marvellous educator and I can thoroughly recommend this book. ISBN 0-8014-8854-0

Microbiology A photographic atlas for the laboratory. Intended for the lab worker but useful for the layman when looking down the microscope at prepared slides of different species of bacteria. Some aspects of lab work are also discussed from different tests ( decarboxylase & gelatin) and identification methods using automated methods which are crucial in a clinical environment.There is also a section on water and soil microbiology. With 400 good quality photographs this is again an excellent book to read. Steve K. Alexander. Dennis Strete. ISBN 0-8053-2732-0

Microbiology perspectives. A photographic survey of the microbial world. George Wistreich. Superb book to have in the laboratory with hundreds of photographs and also gives a historical perspective to the subject. The book also conveys the meaning through photographs of what it means to contract some of these debilitating diseases. ISBN 0-13-856824-3

Garden Of Microbial Delights. Lynn Margulis & Dorian Sagan. If you only buy one book then this should be the one. The book gives an excellent account of the lives of microbes from Viruses, bacteria, protoctists and fungi with some very good drawings to illustrate the accompanying text. ISBN 0-8403-8529-3

Early life. Lynn Margulis & Michael F. Dolan. (Second Edition) This book is a bit more advanced but because Lynn Margulis is such a terrific educator it is still accessible to the majority of readers interested in how life originated on the primitive earth. Professor Margulis and Professor Dolan take us on a 4.5 billion year journey to the very beginning's of the earth to the present day. By numerous digrams and tables along with excellent text they show us how life would adapt to the many different scenarios that it would face during the billions of years to come. ISBN 0-7637-1463-1