1. Observing Microorganisms…………………………………………4

• A Few Time Honored Techniques in Microbiology
• Some Simple Demonstrations

1. Macroscopic Examination....................................................……………12

a. Introduction………………………………………………………….12

1. Protocol…………………………………………………………….13
2. Some Related Information………………………………………….14

• Gram-Positve and Gram-Negative Bacteria
• Media and Reagents
• Selective Growth Media
• Algorithm for Characterizing Unknown Microorganisms
• Characterization of Colony Morphology
• Applications to Identifying Pathogenic Contaminants

2. Microscopic Examination....................................................…………….23

a. Microscopy…………………………………………………………..23

b. Microscopic Examination of Bacteria and Yeast…………………....24

c. Microscopic Examination of Bacteria and Yeast Using Stains………25

• Gram stain………………………………………………………25
• Staining capsulated bacteria……………………………………26
• Staining spore-forming bacteria………………………………..27

Although microorganisms are present in or on nearly everything, it is usually not possible to demonstrate their presence by direct microscopic observation unless their density is high. However, if sterile culture media are exposed to air or inoculated with substances such as soil or lake water, a variety of microorganisms will multiply in the media and can be examined subsequently. To prove that microorganisms are in or on a substance, it is necessary that all media and equipment used by sterile and aseptic technique be employed in performing inoculations and transfers.

The following procedures are meant to demonstrate colony formation by microbial cells inoculated into a petri dish medium. Each cell which can utilize the medium as a source of nutrients and can tolerated the physical conditions present (temperature, pH, atmosphere, etc.) should multiply, resulting, during incubation, in a visible colony of like cells. Different-appearing colonies imply different species of microorganisms; colony appearance is often used in the characterization of bacterial species. When we observe colonies, we cannot assume each arose from just one cell originally planted on the medium, however. A pair, chain or cluster of cells or individual cells which "land" on the medium in close roximity to each other can multiply and produce a single colony. Thus, we use the term colony-forming unit when we consider the common origin for the cells of any colony.

Another term we will often use is culture which is simply a large population of living cells. Examples include a colony (above), a flask of organisms in a liquid medium, and a tube of slanted agar medium on which organisms are growing. A culture of cells, dividing every 20 minutes, can begin with one "new" cell and result in 16,777,216 (i.e. 2²⁴) cells after just 8 hours! A pure culture is composed of identical cells (except for the occasional mutants), possibly have arisen from one cell. A mixed culture contains two or more different kinds of organisms. (We often refer to "young" and "old" cultures, depending on how long they have been incubating since inoculation. We do not, however, refer to "young" and "old" individual cells in the same way, as the cells of most of the bacterial species we work with undergo division every 15 – 30 minutes. Thus, an old cell" --- just about to divide into two "brand new" cells --- may be less than a half hour in age!)

OBSERVING MICROORGANISMS

LB medium:

1. Wipe down your work area with 70% alcohol or some other suitable disinfectant.
2. Use the marker pen to record whatever identifying information you think is necessary on the bottom of the plate.
3. Incubate plates by placing them in an inverted position (so that moisture collecting on the top lid won’t drip down on the developing colonies.
4. Disposable items (pipet tips, cotton swabs, etc.) must be discarded in a biohazard container or into disinfectant (who knows for sure if we’re not picking up pathogens inadvertantly?)
5. Rinse all glassware prior to its washing and sterilization.

Materials (per lab station)

1. Apply disinfectant (e.g. 70% ethanol) to working area.

2. Use permanent marker to label bottom of each agar plate with your initials, date, strain and medium. (e.g. MM294 on LB agar and L /AMP; MM294/pAMP on LB agar and LB/AMP)

3. Flame loop until it glows red hot at tip and several cm. down shaft. Partially lift lid of MM294 culture plate and cool loop by briefly stabbing it into agar in a region distant from the culture streak.

4. Scrape up visible amount of cells from a colony or streak, taking care not to gouge the agar. Replace lid (do not place on bench).

5. Streak LB agar plate with loop according to the following scheme, resterilizing the loop between each streak:

a. in each sector:

Materials (per lab station)

1. Pipette, aseptically with a mechanical pipette, 10 ml broth into flask.

2. Flame loop, cool, and scrape visible amount of cells from plate and transfer to flask. Agitate

3. Incubate overnight in water bath shaker set at 37oC (bacteria), 30oC (yeast).

2. Is shaking necessary? Why or why not?

3. Approximately how many cells are in the culture by the next morning?

4. What physiological state is the culture in by the next morning?

1 YED plate with 50-100 S.cerevisiae colonies (mixed: HA0, HA1, HAT0

2 LB agar plates

2 LB/AMP plates

2 LB/KAN plates

2 YED plates

2 YEAD plates

2 MV plates

replica plate grids

sterile toothpicks

replica plating device ("piston" plus sterile velvet pads)

1. Method 1

2. Method 2:

/

INTRODUCTION

Bacteria represent the most abundant and genetically diverse domain in the living world. We know of the existence of several thousand species of bacteria, but their taxonomy is still incomplete. They are a remarkable group of organisms; their simplicity of structure allows them to have a wide range of biochemical mechanisms which they employ in order to live and break down different nutrients. They are extremely adaptable, and can be ‘trained’ to use substances which appear to be toxic to them on the first exposure.

In nature, it is uncommon to encounter pure cultures. Therefore, when studying bacteria, steps must be taken to transfer individual colonies until one is sure that they are not contaminated. once a pure culture has been obtained, bacteria may then be classified by:

morphology and staining

presence and position of spores

oxygen requirements (obligate/facultative anaerobe)

method of carbohydrate hydrolysis

ability to hydrolyze large molecules (e.g. starch, fats, and proteins by producing externally-acting enzymes)

The purpose of this exercise is to demonstrate of some of the macroscopic properties of a few well-known bacteria and a very well-known fungus; all are harmless. The point of the exercise is to show what colonies of microorganisms of separate genera and species look like and how their growth is influenced by the nutrient supply; and we shall use some microscopic identification protocols that use common staining procedures to identify microorganisms by their cellular morphology.

Even if you do not use these in your classes, a discussion of how this is done is useful since it can dispel the "magic" surrounding identification of microorganisms.....for industrial, medical, epidemiological, etc. purposes. You will be able to demonstrate that microorganisms are classified according to their morphology, nutrition, ability to accept stains, and their biochemical abilities.

A few definitions of terms we shall use follow. The word medium is used to describe the combination of substances on which a particular microbe will grow. Examples of different types of media include:

enrichment media: these are media which encourage the growth of a certain type of microbe (e.g. soil bacteria are plated on media containing a higher concentration of minerals than normal media; for growth of microbes that are able to break down lactose, cells are plated onto media containing lactose as an energy source)

selective media: these are media that suppress some types of microbes, but allow slower-growing varieties to develop (e.g. addition of an antibiotic inhibits growth of bacteria and allows slower-growing fungi to be cultured).

differential media: these media allow a range of biochemical reactions to be seen, from which patterns of biochemical capability may be deduced (e.g. fermentation of different sugars can distinguish among bacteria having different metabolic capabilities; hydrolysis of starch present in the agar indicates secretion of amylase).

The first two groups of media are used to assist in isolating a pure culture; the third, to demonstrate some differences within the microbial group called bacteria. In this exercise, you will experiment with all three.

1. You will be given a number of petri plates. These plates contain growth medium mixed with agar to provide a solid growth medium. Cultures of several different kinds of microorganisms were grown. They contain different genera and species of bacteria and a fungus (yeast). The cultures were diluted, spread on the different solid growth media, and incubated at the organism’s optimal growth temperature (30oC for yeast; 37oC for bacteria) for one (bacteria) or 2-3 days (yeast).

2. Note for each of the organisms such things as:

growth or no growth (+++, ++, +, -), color of colonies, colony morphology (size, smooth or rough looking, contained or spread out; and any other distinguishing characteristics you think help differentiate one from another)

B.subtilis LB

B.cereus LB + glucose

M.luteus LB + ampicillin

S.epidermidis NA

E.faecalis NA + starch

L. plantarum Minimal (with glucose)

K.pneumoniae MAC

S.marcescens EMB

P. fluourescens glucose/lactose fermentation broth

S.cerevisiae motility agar medium

tryptone broth

nitrate broth

This supplementary material was taken from several sources: the UW-Madison Bacteriology 304 Laboratory Manual for Spring, 1988 (Dr. Tim Paustian), General Microbiology: A Laboratory Manual (John Lindquist), and from the text, Microbiology (by Dulbecco,et al). You are strongly encouraged to read this over, along with the material included in Section III from this manual. It is most likely much more technical than you would need or use in your classes, but it also gives you a window on how the science of bacteriology is done and the algorithms used in the practical applications of this science in, for example, medical diagnosis, analysis of samples for contaminating organisms, etc. It can provide for you the basis for constructing a protocol to isolate and characterize microorganisms from "real world" sample

GRAM NEGATIVE ORGANISMS

Escherichia coli: Short rod, often oval-shaped; facultatively anaerobic. Part of the normal flora of the intestinal tract of warm-blooded animals and not found free-living in nature (thus, a good indicator organism for fecal contamination.) Generally non-pathogenic although some strains may cause severe food-borne illness; may be involved in urinary tract and other infections.

Klebsiella pneumoniae: Short rod, often oval-shaped; facultatively anaerobic. This organism may appear in gram-stained preparations as a mixture of gram-negative and positive cells. Found free-living in soil and on leaves of plants, also in the respiratory and intestinal tract of animals on occasion. Many strains fix atmospheric nitrogen. Often heavily encapsulated, producing mucoid (slimy) colonies. Like all other species of the genus, it is non-motile. Pathogenic strains of this species are a major cause of bacterial pneumonia in humans.

Serratia marcescens: Short rod, often oval-shaped; facultatively anaerobic. This organism is often found in soil and water where it is involved in the decomposition of organic matter by various extracellular enzymes, including enzymes which degrade DNA and chitin. It has been found (among many other organisms) in the water which collects in the leaves of the pitcher plant where it is involved in the breakdown of protein and chitin from entrapped insects. Some strains produce an orange-red pigment, prodigiosin, in a temperature-dependent manner.

Pseudomonas fluorescens: Rod, strictly aerobic. Common soil and water organism, often involved in the spoilage of meat and dairy products. Produces diffusible, fluorescent siderphore, fluorescin. Most strains of this species reduce nitrate to nitrite; some can reduce it to nitrogen gas. Related to the severe pathogen, P. aeruginosa.

Microcococcus luteus: Coccus, strictly aerobic. Cells generall in small clusters or in a sarcina arrangement. Common airborne contaminant of culture media. Usually produces a bright, yellow pigment.

Staphylococcus epidermidis: Coccus, facultatively anaerobic. Cells usually in staphylococcal arrangement. Common, non-pathogenic organism found on human skin. Related to the pathogen, S. aureus.

Enterococcus faecalis: Coccus, aerotolerant anaerobe. Cells generally in short chains or pairs. Cells are often oval-shaped; may resemble some of the above gram-negative bacteria if overdecolorized in the gram stain procedure. Part of the normal, non-pathogenic flora of the human and mammalian intestine; often found free-living in soil and on plants.

Lactobacillus plantarum: Rod (short to long), aerotolerant anaerobe. Found associated with material of plant origin; used in the natural fermentation of sauerkraut and pickles.

Bacillus subtilis: Rod, generally considered to be strictly aerobic although may show very weak aneaerobic growth with glucose. Found in soil; active in breaking down plant and animal matter. Tends to be gram-variable. Produces endospores: as nutrients become depleted in a culture, an endospore forms inside the cell, often becoming liberated and observable as free endospores.

Bacillus cereus: Large rod, facultatively anaerobic. Gram variable and forms endospores. Commonly found in soil and on dry vegetable matter; may cause a food-borned illness if ingested in large numbers. Closely related to the anthrax-causing pathogen, B. anthracis.

Agar. Agar is used as a solidifying agent in media. It is an extract of marine algae and contains a complex polysaccharide (agarose) as its main ingredient, but also contains many impurities. These contaminants do not interfere with most micorbioogical experiments. Agar solutions melt at 100°C and solidify at 43°C. Most microorganisms cannot use agar as a nutrient.

Body Fluids. Pathogens often have complex and unknown growth requirements. These growth requirements are often satisfied by adding whole blood or defibrinated blood, serum, plasma, or other bodily fluids to the culture media. Bodily fluids may also contain substances that detoxify inhibitory compounds in the medium.

Buffers. Microorganisms sometimes produce organic acids and/or bases as waste products during growth, which can alter the pH of the medium. When necessary, compounds are added to culture medium to maintain an optimum pH range for the microorganism in question. Common buffers include; sodium phosphates, potassium phosphates, and peptones.

Extracts. Animal or plant tissues (beef, liver, yeast, etc.) are extracted by boiling and then concentrated to a paste or dried. Extracts contribute necessary amino acids, vitamins and coenzymes that support the growth of fastidious microorganisms. They are frequently used in culture media as a substitute for fresh infusions.

Infusions. Infusions are aqueous extracts of ground, defatted meat (muscle, liver, heart, brain, etc.) prepared by infusing meat in hot or cold water, then boiling or steaming them. Many have a high content of vitamins, minerals, amino acids and other nitrogen compounds, and some sugar is present.

Peptones. These complex mixtures of organic and inorganic compounds are obtained by digestion of proteinaceous animal or plant materials (meat scraps, beef muscle, gelatin, milk casein, soybean meal, etc.). Peptones are rich in nitrogen sources (peptides, amino acids, nitrogen bases, etc.) and they contain a great variety of organic and inorganic materials, but they may be deficient in certain minerals or vitamins. Several different types of commercially-prepared peptones are available for microbiological purposes. Some examples are: Trypticase or Tryptone (Pancreatic digestion of milk casein), Phytone (papaic digest of soya meal) and Peptone (a digest of beef muscle). Peptones find use in conjunction with infusions for cultivation of fastidious organisms. Peptones supply the necessary amino acids and polypeptides, while infusions supply vitamins, coenzymes and minerals as well as additional nitrogen compounds.

pH Indicators. An acid-base indicator is often added to differential media to detect changes in hydrogen ion concentration during the growth of an organism. These colored dyes (brom cresol purple, bromthymol blue and phenol red as a few examples) will change color as the pH changes. pH indicators can be found in carbohydrate fermentation broth, Simmons citrate agar, MacConkey’s Agar, Glucose O/F medium, Kliger’s Iron Agar and many others.

Reducing Agents. Strictly anaerobic and microaerophillic organisms cannot grow optimally in the presence of atmospheric concentrations of oxygen. Certain chemicals can be added to media to reduce the oxidation-reduction potential and therefore the oxygen concentration. Cystine and thioglycollate are common reducing agents added to certain culture media (e.g., thioglycollate medium) to reduce the oxidation reduction potential.

Selective Agents. It is often desirable to select for a specific group of microorganisms (Gram negative, antibiotic resistant, etc.) Selective media may contain antimicrobial agents such as crystal violet, bile salts, sodium azide, potassium tellurite, or antibiotics to suppress or inhibit the growth of one type of organism but allow the growth of another.

YED: A rich medium used in culturing yeast

yeast extract (10.0g/L)

glucose (20.0 g/L)

agar (20 g/L)

Luria-Bertani (LB): A rich medium used in molecular biology. Glucose can be added (2.0 g/L) for

tryptone (10.0g/L)

yeast extract (5.0 g/L)

NaCl (10.0 g/L)

agar (15.0 g/L)

pH 7.0

Nutrient Agar (NA): An all-purpose medium for organisms which are not nutritionally fastidious. Soluble

starch added (4.0 g/L) to test for the production of extracelluar amylases.

beef extract (5.0 g/L)

peptone (3.0 g/L)

agar (15.0 g/L)

pH 6.8

Minimal Medium: For experiments when a chemically defined medium is required; carbon source can vary.

ammonium sulfate (1.0 g/L)

dipotassium phosphate (7.0 g/L)

monopotassium phosphate (3.0 g/L)

magnesium sulfate (0.1 g/L)

carbon source (1-10 g/L)

agar (15.0 g/L)

pH 7

MaConkeys: For the isolation and differentiation of gram-negative bacteria including the enterics.

peptone (17.0 g/L)

proteose peptone (3.0 g/L)

lactose (10.0 g/L)

bile salts (1.5 g/L)

neutral red (0.03 g/L)

crystal violet (0.001 g/L)

agar (13.5 g/L)

pH 7.1

Eosin Methylene Blue (EMB): For the isolation and detection of gram-negative bacteria. Colonies of

peptone (1.0. g/L)

lactose (10.0g/L)

dipotassium phosphate (2.0 g/L)

eosin Y (0.40 g/L)

methylene blue (0.065 g/L)

agar (15.0 g/L)

pH 7.1

glucose/lactose fermentation broth

catalase test

motility

tryptone degradation to indole

nitrate reduction

• Carefully layer half a dropperful of Kovac's reagent onto the surface of the tryptone broth cultures and let sit for a few minutes. The development of a red ring at the broth reagent interface is a positive test for indole production. (Note that only one of the known cultures is expected to give a positive test for indole.)

.CHARACTERISTICS OF COLONY MORPHOLOGY

As microorganisms grow and divide on solid surfaces they form specific patterns of growth. Individual cells (or a few identical cells) will continue to grow and divide and form discrete units called colonies, the morphology of which is characteristic of that microbial species. An accurate description of an isolated colony can greatly aid in the identification of the microorganism. Microbiologists have developed standard words with specific meanings to describe colony form, elevation and margin (see Figure below). Scientists use this jargon to help accurately communicate their observations. This is a common theme in all disciplines of science. Imagine what would happen if everyone used different terminologies? There would be no effective communication between scientist.

The Enterobacteriaciae are a large group of Gram negative, non-sporulating, short rods that are mobile or non-mobile. They have relatively simple nutritional requ;irements and are facultative anaerobes that ferment glucose to acid under anaerobic conditions. Many of them can be islated from the intestinal tract and the term "enterics" has come to be synonymous with this groups --- even though most are not limited to this environment. Because of the medical importance of many of the members of this group, it is probably the best characterized collection of prokaryotes.

Shigella is a close relative of E. coli and most isolates are pathogenic. This organism causes a diarrheal illness that involves colonization and attack of the intestines. One to four days after ingestion the victim will experience a sudden onset of abdominal cramps, fever and profuse bloody diarrhea containing mucous. The illness normally lasts a few days and can be caused by as few as 100 organisms. Improperly prepared food and contaminated water are typical sources of infection with Shigella.

Salmonella is a genus of pathogenic organisms infecting humans and many mammals, birds and reptiles. Organisms in this genus are so genetically similar that they are now considered as belonging to two species – S. bongori and S. choleraesuis. There are at least seven, genetically-distinct subgroups and over 2500 serovars (formerly called serotypes). Each serovar is distinguished by a unique combination of cell wall and flagellar antigens. Serovar recognition is important in epidemiology; an outbreak or epidemic caused by organisms of one serovar can often be traced to a common source. Decades ago, it was the fashion to consider each serovar a species. Today, most serovars are given names which are designated as a species names such as Salmonella typhimurium, S. enteritidis and S. heidelberg , but this is only for convenience not taxonomic accuracy. These three serovars are responsible for about half of the cases of human Salmonella infection in the United States.

Most serovars of Salmonella cause gastroenteritis of varying degrees or severity, with or without bacteremia. The source of gastroenteritis is usually contaminated food containing >106 cells/g (or ml). Symptoms generally appear in 8 to 30 hours after ingestion and include nausea, fever, diarrhea, and abdominal pain. Symptoms usually subside in one to two days. Certain host-adapted, biochemically-distinct serovars ("bioserovars") cause life-threatening illnesses such as typhoid fever by S. typhi, "hog cholera" by S. cholerae-suis (also pathogenic to humans) and "fowl typhoid" by S. gallinarum.

Organisms of the genus Edwardsiella are known to cause disease in humans and a variety of warm and cold-blooded vertebrates. E. tarda is an occasional opportunistic pathogen for humans, causing wound infections and, in less-industrialized countries, gastroenteritis. E. tarda and E. ictaluri have caused massive infections of commercially-raised catfish with considerable economic loss.

The genus Yersinia is an infamous member of the enterics. Y. enterololitica is involved in many cases of gastroenteritis. Y. pestis is the cause of bubonic plague, a dreaded disease until relatively recently. In the fourteenth century the black death, as it was called, claimed the lives of one-third of Europe's population. The disease, in some forms, can kill in a matter of hours. Bubonic plague is harbored in rats and is spread to humans by fleas. Due to good sanitation (a low population of rats) a recurrence of this disease on a large scale in the U. S. is unlikely, hopefully.

Proteus, Providencia and Morganella. This group of non-pathogenic organisms is distinguished by the ability to deaminate phenylalanine. They are often implicated in spoilage of fish and seafood. Proteus forms unusual swarming patterns on agar plates that have a concentric ring shape. This unusual pattern is caused by alternating phases of rapid motility and less motile growth.

Citrobacter is a commonly isolated, non-pathogenic enteric. It is of interest because it frequently gives false positives in tests designed to detect the presence of Salmonella in food.

Erwinia species are pathogenic for plants causing various soft rots and wilts of important crops and vegetables. If you have ever thrown away soft rotten potatoes, they were probably attacked by E. carotovora, the cause of potato soft-rot.

Occasional strains of Serratia form red pigmented colonies on agar plates. The red color is due to the synthesis of a series of linear tripyroles (prodigiosins). The pigment is curious in that it bears resemblance to the tetrapyroles involved in energy transfers (i.e. hemes, cytochromes, chlorophylls, etc.). The function of the prodigiosins, however is unknown.

Coliforms. A coliform is defined as a non-sporeforming, facultatively anaerobic, Gram negative rod, which ferments lactose to acid and gas within 48 hours at 35°C. This is an operational definition used in water analysis (see below) and any organism isolated meeting these requirements is a coliform. In practice, isolated coliforms are almost always Enterobacteriaceae from the genera Enterobacter, Klebsiella, and Escherichia (also lactose positive strains of Citrobacter).

Klebsiella are of interest due to the ability of most strains to use N2 as sole nitrogen source. Study of this organism has helped to unlock many of the mysteries of nitrogen fixation (an agronomically important process). K. pneumoniae is a human pathogen sometimes causing pneumonia. Enterobacter and Klebsiella are widely distributed in water, soil, and plant material.

One member of the genus Escherichia, E. coli, is the "star" organism of biology. Much of what we know about biological processes, in general, have come from studies of E. coli. It is also useful as a tool for harboring and amplifying DNA in genetic engineering. This organism's main habitat is the intestinal tract of warm blooded animals, but it can also be found in environments contaminated with feces. Some strains of E. coli also cause gastrointestinal disease. Several recent severe outbreaks have been traced to undercooked meat infected with pathogenic E. coli.

Since it is particularly important to assure a safe water supply, it is particularly critical to monitor for the presence of these pathogens. However, it would be expensive and time consuming to check for all the them; so, an indicator organism is used to assy for fecal contamination. An indicator organism must have four properties to be useful for water analysis.

• The only natural environment of the microbe should be in association with feces and it should be always present.
• It should not grow outside of its natural environment.
• The bacterium should not survive longer than the most viable pathogen, but not so long so that historical events are detected.
• It should be easy to detect.

Coliforms come close to fulfiling all these criteria and are the standard indicator orgnisms used to test for the biological pollution of water.

Enterics are very similar to each other, physiologically; but the method they use to ferment glucose is an important difference that can be used to distinguish among them. For example, some metabolize glucose to give lactic, succinic, acetic, or formic acid. The large amount of acid produced lowers the pH of the medium (below 4.4) and can be detected by suitable pH sensitive dye indicators added to the medium. Others break down glucose to products that also include the neutral butanediol. In general, Enterics:

• grow readily on ordinary media under aerobic or anaerobic conditions
• (most) will grow in simple synthetic medium, often with a single carbon source
• utilize glucosefermentativly to produce acid or acid plus gas
• reduce nitrates to nitrite
• give negative oxidase test
• can usually be distinguished according to genus and species by:
• capacity to ferment specific carbohydrates (e.g. lactose)
• ability to use certain substrates as sole carbon source
• give rise to characteristic end products

Below are the differential criteria one uses to characterize members of this group.

1 Positive reaction may be weak.

2 76-89% are positive

3 May show equivocal (orange) reaction.

In this exercise, we wish to demonstrate how the ingenious use of selective medium can distinguish among several differential characteristics of enterics in a single culture. We have plated out several of the strains on Kliger’s Iron Agar. This is a rich medium that is poured into slants:

Most of you have had experience using microscopes and most likely use them in your classes. As you know, microscopes are neither simple nor direct extensions of your eyes; they are experimental tools with specific strengths and limitations . And you also know that the quality of information we obtain with a microscope depends of several factors which include the quality of the optical system, the quality of the preparation and the quality of the observations made by the person using the microscope.

The maximum resolution of a light microscope is achieved with visible light of the smallest wavelength and an objective with the largest numerical aperture Your microscope may be able to resolve discrete images of objects separated by as little as 0.2µm; but this fine resolution is of no use to you unless the resolved image is enlarged to the point where this detail is visible to your eyes (with resolution about 100µm). So the magnification must be of the order 100/0.2 = 500x. The total magnification for your microscope can be calculated by multiplying the ocular magnification by the objective magnification.

• bright field is the system used to view stained or naturally pigmented specimens (i.e the stains or pigments absorb or alter the light that passes through the specimen); the condenser diaphragm on the microscope can be adjusted to enhance contrast by decreasing intensity of the background light relative to the object on the slide .

phase contrast is invaluable for most structures in living tissues since they are unpigmented and so are essentially invisible under bright field microscopy. They do, however, tend to differ in refractive index. The phase contrast system converts differences in refractive index into differences in light intensity. The "contrast" is generated by separating and adjusting the phase relationships of direct light (passing through unchanged) and diffracted light (scattered by the specimen)

MICROSCOPIC EXAMINATION OF BACTERIA AND YEAST

You will be given cultures of the following microorganisms (both grown in liquid and solid medium):

Put a drop or two of each culture on a slide, cover with a coverslip, and observe under bright field using all objectives, including oil immersion, and phase contrast. Record your observations (e.g. under what conditions were you able to visualize the cells? were you able to distinguish one cell from another? were you able to see any internal structure? would you be able to classify the organisms according to the diagram below of bacterial forms?) Now add diluted methylene blue and observe each sample; record your observations

1. Gram Stain

Among pathogenic bacteria, Gram negative species are generally more threatening than Gram positive. Since Gram negative bacteria contain more complex cell walls, they are generally more resistant to antibiotics (note that this does NOT mean that antibiotic resistance develops in otherwise sensitive bacteria by cells acquiring the ability to manufacture a complex cell wall)

1. crystal violet

2. iodine

3. alcohol

4. safranine