Genetic Engineering, history and future Altering the Face of Science

Science is a creature that continues to evolve at a much higher rate than the beings that gave it birth. The transformation time from tree-shrew, to ape, to human far exceeds the time from analytical engine, to calculator, to computer. But science, in the past, has always remained distant. It has allowed for advances in production, transportation, and even entertainment, but never in history will science be able to so deeply affect our lives as genetic engineering will undoubtedly do.

With the birth of this new technology, scientific extremists and anti-technologists have risen in arms to block its budding future. Spreading fear by misinterpretation of facts, they promote their hidden agendas in the halls of the United States congress. Genetic engineering is a safe and powerful tool that will yield unprecedented results, specifically in the field of medicine. It will usher in a world where gene defects, bacterial disease, and even aging are a thing of the past.

By understanding genetic engineering and its history, discovering its possibilities, and answering the moral and safety questions it brings forth, the blanket of fear covering this remarkable technical miracle can be lifted. The first step to understanding genetic engineering, and embracing its possibilities for society, is to obtain a rough knowledge base of its history and method. The basis for altering the evolutionary process is dependant on the understanding of how individuals pass on characteristics to their offspring.

Genetics achieved its first foothold on the secrets of nature’s evolutionary process when an Austrian monk named Gregor Mendel developed the first “laws of heredity. ” Using these laws, scientists studied the characteristics of organisms for most of the next one hundred years following Mendel’s discovery. These early studies concluded that each organism has two sets of character determinants, or genes (Stableford 16). For instance, in regards to eye color, a child could receive one set of genes from his father that were encoded one blue, and the other brown.

The same child could also receive two brown genes from his mother. The conclusion for this inheritance would be the child has a three in four chance of having brown eyes, and a one in three chance of having blue eyes (Stableford 16). Genes are transmitted through chromosomes which reside in the nucleus of every living organism’s cells. Each chromosome is made up of fine strands of deoxyribonucleic acids, or DNA. The information carried on the DNA determines the cells function within the organism.

Sex cells are the only cells that contain a complete DNA map of the organism, therefore, “the structure of a DNA molecule or combination of DNA molecules determines the shape, form, and function of the [organism’s] offspring ” (Lewin 1). DNA discovery is attributed to the research of three scientists, Francis Crick, Maurice Wilkins, and James Dewey Watson in 1951. They were all later accredited with the Nobel Price in physiology and medicine in 1962 (Lewin 1). “The new science of genetic engineering aims to take a dramatic short cut in the slow process of evolution” (Stableford 25).

In essence, scientists aim to remove one gene from an organism’s DNA, and place it into the DNA of another organism. This would create a new DNA strand, full of new encoded instructions; a strand that would have taken Mother Nature millions of years of natural selection to develop. Isolating and removing a desired gene from a DNA strand involves many different tools. DNA can be broken up by exposing it to ultra-high-frequency sound waves, but this is an extremely inaccurate way of isolating a desirable DNA section (Stableford 26).

A more accurate way of DNA splicing is the use of “restriction enzymes, which are produced by various species of bacteria” (Clarke 1). The restriction enzymes cut the DNA strand at a particular location called a nucleotide base, which makes up a DNA molecule. Now that the desired portion of the DNA is cut out, it can be joined to another strand of DNA by using enzymes called ligases. The final important step in the creation of a new DNA strand is giving it the ability to self-replicate.

This can be accomplished by using special pieces of DNA, called vectors, that permit the generation of multiple copies of a total DNA strand and fusing it to the newly created DNA structure. Another newly developed method, called polymerase chain reaction, allows for faster replication of DNA strands and does not require the use of vectors (Clarke 1). The possibilities of genetic engineering are endless. Once the power to control the instructions, given to a single cell, are mastered anything can be accomplished.

For example, insulin can be created and grown in large quantities by using an inexpensive gene manipulation method of growing a certain bacteria. This supply of insulin is also not dependant on the supply of pancreatic tissue from animals. Recombinant factor VIII, the blood clotting agent missing in people suffering from hemophilia, can also be created by genetic engineering. Virtually all people who were treated with factor VIII before 1985 acquired HIV, and later AIDS.

Being completely pure, the bioengineered version of factor VIII eliminates any possibility of viral infection. Other uses of genetic engineering include creating disease resistant crops, formulating milk from cows already containing pharmaceutical compounds, generating vaccines, and altering livestock traits (Clarke 1). In the not so distant future, genetic engineering will become a principal player in fighting genetic, bacterial, and viral disease, along with controlling aging, and providing replaceable parts for humans. Medicine has seen many new innovations in its history.

The discovery of anesthetics permitted the birth of modern surgery, while the production of antibiotics in the 1920s minimized the threat from diseases such as pneumonia, tuberculosis and cholera. The creation of serums which build up the bodies immune system to specific infections, before being laid low with them, has also enhanced modern medicine greatly (Stableford 59). All of these discoveries, however, will fall under the broad shadow of genetic engineering when it reaches its apex in the medical community. Many people suffer from genetic diseases ranging from thousands of types of cancers, to blood, liver, and lung disorders.

Amazingly, all of these will be able to be treated by genetic engineering, specifically, gene therapy. The basis of gene therapy is to supply a functional gene to cells lacking that particular function, thus correcting the genetic disorder or disease. There are two main categories of gene therapy: germ line therapy, or altering of sperm and egg cells, and somatic cell therapy, which is much like an organ transplant. Germ line therapy results in a permanent change for the entire organism, and its future offspring. Unfortunately, germ line therapy, is not readily in use on humans for ethical reasons.

However, this genetic method could, in the future, solve many genetic birth defects such as downs syndrome. Somatic cell therapy deals with the direct treatment of living tissues. Scientists, in a lab, inject the tissues with the correct, functioning gene and then re-administer them to the patient, correcting the problem (Clarke 1). Along with altering the cells of living tissues, genetic engineering has also proven extremely helpful in the alteration of bacterial genes. Transforming bacterial cells is easier than transforming the cells of complex organisms” (Stableford 34).

Two reasons are evident for this ease of manipulation: DNA enters, and functions easily in bacteria, and the transformed bacteria cells can be easily selected out from the untransformed ones. Bacterial bioengineering has many uses in our society, it can produce synthetic insulins, a growth hormone for the treatment of dwarfism and interferons for treatment of cancers and viral diseases (Stableford 34). Throughout the centuries disease has plagued the world, forcing everyone to take part in a virtual “lottery with the agents of death” (Stableford 59).

Whether viral or bacterial in nature, such disease are currently combated with the application of vaccines and antibiotics. These treatments, however, contain many unsolved problems. The difficulty with applying antibiotics to destroy bacteria is that natural selection allows for the mutation of bacteria cells, sometimes resulting in mutant bacterium which is resistant to a particular antibiotic. This now indestructible bacterial pestilence wages havoc on the human body. Genetic engineering is conquering this medical dilemma by utilizing diseases that target bacterial organisms.

These diseases are viruses, named bacteriophages, “which can be produced to attack specific disease-causing bacteria” (Stableford 61). Much success has already been obtained by treating animals with a “phage” designed to attack the E. coli bacteria (Stableford 60). Diseases caused by viruses are much more difficult to control than those caused by bacteria. Viruses are not whole organisms, as bacteria are, and reproduce by hijacking the mechanisms of other cells. Therefore, any treatment designed to stop the virus itself, will also stop the functioning of its host cell.

A virus invades a host cell by piercing it at a site called a “receptor”. Upon attachment, the virus injects its DNA into the cell, coding it to reproduce more of the virus. After the virus is replicated millions of times over, the cell bursts and the new viruses are released to continue the cycle. The body’s natural defense against such cell invasion is to release certain proteins, called antigens, which “plug up” the receptor sites on healthy cells. This causes the foreign virus to not have a docking point on the cell.

This process, however, is slow and not effective against a new viral attack. Genetic engineering is improving the body’s defenses by creating pure antigens, or antibodies, in the lab for injection upon infection with a viral disease. This pure, concentrated antibody halts the symptoms of such a disease until the bodies natural defenses catch up. Future procedures may alter the very DNA of human cells, causing them to produce interferons. These interferons would allow the cell to be able determine if a foreign body bonding with it is healthy or a virus.

In effect, every cell would be able to recognize every type of virus and be immune to them all (Stableford 61). Current medical capabilities allow for the transplant of human organs, and even mechanical portions of some, such as the battery powered pacemaker. Current science can even re-apply fingers after they have been cut off in accidents, or attach synthetic arms and legs to allow patients to function normally in society. But would not it be incredibly convenient if the human body could simply regrow what it needed, such as a new kidney or arm?

Genetic engineering can make this a reality. Currently in the world, a single plant cell can differentiate into all the components of an original, complex organism. Certain types of salamanders can re-grow lost limbs, and some lizards can shed their tails when attacked and later grow them again. Evidence of regeneration is all around and the science of genetic engineering is slowly mastering its techniques. Regeneration in mammals is essentially a kind of “controlled cancer”, called a blastema.

The cancer is deliberately formed at the regeneration site and then converted into a structure of functional tissues. But before controlling the blastema is possible, “a detailed knowledge of the switching process by means of which the genes in the cell nucleus are selectively activated and deactivated” is needed (Stableford 90). To obtain proof that such a procedure is possible one only needs to examine an early embryo and realize that it knows whether to turn itself into an ostrich or a human.

After learning the procedure to control and activate such regeneration, genetic engineering will be able to conquer such ailments as Parkinson’s, Alzheimer’s, and other crippling diseases without grafting in new tissues. The broader scope of this technique would allow the re-growth of lost limbs, repairing any damaged organs internally, and the production of spare organs by growing them externally (Stableford 90). Ever since biblical times the lifespan of a human being has been pegged at roughly 70 years.

But is this number truly finite? In order to uncover the answer, knowledge of the process of aging is needed. A common conception is that the human body contains an internal biological clock which continues to tick for about 70 years, then stops. An alternate “watch” analogy could be that the human body contains a certain type of alarm clock, and after so many years, the alarm sounds and deterioration beings. With that frame of thinking, the human body does not begin to age until a particular switch is tripped.

In essence, stopping this process would simply involve a means of never allowing the switch to be tripped. W. Donner Denckla, of the Roche Institute of Molecular Biology, proposes the alarm clock theory is true. He provides evidence for this statement by examining the similarities between normal aging and the symptoms of a hormonal deficiency disease associated with the thyroid gland. Denckla proposes that as we get older the pituitary gland begins to produce a hormone which blocks the actions of the thyroid hormone, thus causing the body to age and eventually die.

If Denckla’s theory is correct, conquering aging would simply be a process of altering the pituitary’s DNA so it would never be allowed to release the aging hormone. In the years to come, genetic engineering may finally defeat the most unbeatable enemy in the world, time (Stableford 94). The morale and safety questions surrounding genetic engineering currently cause this new science to be cast in a false light. Anti-technologists and political extremists spread false interpretation of facts coupled with statements that genetic engineering is not natural and defies the natural order of things.

The morale question of biotechnology can be answered by studying where the evolution of man is, and where it is leading our society. The safety question can be answered by examining current safety precautions in industry, and past safety records of many bioengineering projects already in place. The evolution of man can be broken up into three basic stages. The first, lasting millions of years, slowly shaped human nature from Homo erectus to Home sapiens. Natural selection provided the means for countless random mutations resulting in the appearance of such human characteristics as hands and feet.

The second stage, after the full development of the human body and mind, saw humans moving from wild foragers to an agriculture based society. Natural selection received a helping hand as man took advantage of random mutations in nature and bred more productive species of plants and animals. The most bountiful wheats were collected and re-planted, and the fastest horses were bred with equally faster horses. Even in our recent history the strongest black male slaves were mated with the hardest working female slaves.

The third stage, still developing today, will not require the chance acquisition of super-mutations in nature. Man will be able to create such super-species without the strict limitations imposed by natural selection. By examining the natural slope of this evolution, the third stage is a natural and inevitable plateau that man will achieve (Stableford 8). This omniscient control of our world may seem completely foreign, but the thought of the Egyptians erecting vast pyramids would have seem strange to Homo erectus as well.

Many claim genetic engineering will cause unseen disasters spiraling our world into chaotic darkness. However, few realize that many safety nets regarding bioengineering are already in effect. The Recombinant DNA Advisory Committee (RAC) was formed under the National Institute of Health to provide guidelines for research on engineered bacteria for industrial use. The RAC has also set very restrictive guidelines requiring Federal approval if research involves pathogenicity (the rare ability of a microbe to cause disease) (Davis, Roche 69). “It is well established that most natural bacteria do not cause disease.

After many years of experimentation, microbiologists have demonstrated that they can engineer bacteria that are just as safe as their natural counterparts” (Davis, Rouche 70). In fact the RAC reports that “there has not been a single case of illness or harm caused by recombinant [engineered] bacteria, and they now are used safely in high school experiments” (Davis, Rouche 69). Scientists have also devised other methods of preventing bacteria from escaping their labs, such as modifying the bacteria so that it will die if it is removed from the laboratory environment.

This creates a shield of complete safety for the outside world. It is also thought that if such bacteria were to escape it would act like smallpox or anthrax and ravage the land. However, laboratory-created organisms are not as competitive as pathogens. Davis and Roche sum it up in extremely laymen’s terms, “no matter how much Frostban you dump on a field, it’s not going to spread” (70). In fact Frostbran, developed by Steven Lindow at the University of California, Berkeley, was sprayed on a test field in 1987 and was proven by a RAC committee to be completely harmless (Thompson 104).

Fear of the unknown has slowed the progress of many scientific discoveries in the past. The thought of man flying or stepping on the moon did not come easy to the average citizens of the world. But the fact remains, they were accepted and are now an everyday occurrence in our lives. Genetic engineering too is in its period of fear and misunderstanding, but like every great discovery in history, it will enjoy its time of realization and come into full use in society.

Starch Hydrolysis Test

Introduction

Starchy substances constitute the major part of the human diet for most of the people in the world, as well as many other animals. They are synthesized naturally in a variety of plants. Some plant examples with high starch content are corn, potato, rice, sorghum, wheat, and cassava. It is no surprise that all of these are part of what we consume to derive carbohydrates. Similar to cellulose, starch molecules are glucose polymers linked together by the alpha-1,4 and alpha-1,6 glucosidic bonds, as opposed to the beta-1,4 glucosidic bonds for cellulose.

In order to make use of the carbon and energy stored in starch, the human digestive system, with the help of the enzyme amylases, must first break down the polymer to smaller assimilable sugars, which is eventually converted to the individual basic glucose units.Because of the existence of two types of linkages, the alpha-1,4 and the alpha-1,6, different structures are possible for starch molecules. An unbranched, single chain polymer of 500 to 2000 glucose subunits with only the alpha-1,4 glucosidic bonds is called amylose. On the other hand, the presence of alpha-1,6 glucosidic linkages results in a branched glucose polymer called amylopectin.

The degree of branching in amylopectin is approximately one per twenty-five glucose units in the unbranched segments. Another closely related compound functioning as the glucose storage in animal cells is called glycogen, which has one branching per 12 glucose units. The degree of branching and the side chain length vary from source to source, but in general the more the chains are branched, the more the starch is soluble.

Starch is generally insoluble in water at room temperature. Because of this, starch in nature is stored in cells as small granules which can be seen under a microscope. Starch granules are quite resistant to penetration by both water and hydrolytic enzymes due to the formation of hydrogen bonds within the same molecule and with other neighboring molecules. However, these inter- and intra-hydrogen bonds can become weak as the temperature of the suspension is raised. When an aqueous suspension of starch is heated, the hydrogen bonds weaken, water is absorbed, and the starch granules swell. This process is commonly called gelatinization because the solution formed has a gelatinous, highly viscous consistency. The same process has long been employed to thicken broth in food preparation.

Depending on the relative location of the bond under attack as counted from the end of the chain, the products of this digestive process are dextrin, maltotriose, maltose, and glucose, etc. Dextrins are shorter, broken starch segments that form as the result of the random hydrolysis of internal glucosidic bonds. A molecule of maltotriose is formed if the third bond from the end of a starch molecule is cleaved; a molecule of maltose is formed if the point of attack is the second bond; a molecule of glucose results if the bond being cleaved is the terminal one; and so on.

As can be seen from the exercises in Experiment No. 3, the initial step in random depolymerization is the splitting of large chains into various smaller sized segments. The breakdown of large particles drastically reduces the viscosity of gelatinized starch solution, resulting in a process called liquefaction because of the thinning of the solution. The final stages of depolymerization are mainly the formation of mono-, di-, and tri-saccharides. This process is called saccharification, due to the formation of saccharides.

Since a wide variety of organisms, including humans, can digest starch, alpha-amylase is obviously widely synthesized in nature, as opposed to cellulase. For example, human saliva and pancreatic secretion contain a large amount of alpha-amylase for starch digestion. The specificity of the bond attacked by alpha-amylases depends on the sources of the enzymes. Currently, two major classes of alpha-amylases are commercially produced through microbial fermentation. Based on the points of attack in the glucose polymer chain, they can be classified into two categories, liquefying and saccharifying.

Because the bacterial alpha-amylase to be used in this experiment randomly attacks only the alpha-1,4 bonds, it belongs to the liquefying category. The hydrolysis reaction catalyzed by this class of enzymes is usually carried out only to the extent that, for example, the starch is rendered soluble enough to allow easy removal from starch-sized fabrics in the textile industry. The paper industry also uses liquefying amylases on the starch used in paper coating where breakage into the smallest glucose subunits is actually undesirable. (One cannot bind cellulose fibers together with sugar!)

On the other hand, the fungal alpha-amylase belongs to the saccharifying category and attacks the second linkage from the nonreducing terminals (i.e. C4 end) of the straight segment, resulting in the splitting off of two glucose units at a time. Of course, the product is a disaccharide called maltose. The bond breakage is thus more extensive in saccharifying enzymes than in liquefying enzymes. The starch chains are literally chopped into small bits and pieces. Finally, the amyloglucosidase (also called glucoamylase) component of an amylase preparation selectively attacks the last bond on the nonreducing terminals. The type to be used in this experiment can act on both the alpha-1,4 and the alpha-1,6 glucosidic linkages at a relative rate of 1:20, resulting in the splitting off of simple glucose units into the solution. Fungal amylase and amyloglucosidase may be used together to convert starch to simple sugars. The practical applications of this type of enzyme mixture include the production of corn syrup and the conversion of cereal mashes to sugars in brewing.

Thus, it is important to specify the source of enzymes when the actions and kinetics of the enzymes are compared. Four types of alpha-amylases from different sources will be employed in this experiment: three of microbial origin and one of human origin. The effects of temperature, pH, substrate concentration, and inhibitor concentration on the kinetics of amylase catalyzed reactions will be studied. Finally, the action of the amylase preparations isolated from microbial sources will be compared to that from human saliva.

Methods

In this demonstration, the action of two bacterial species, Bacillus subtilis and Escherichia coli, is compared on starch agar. After inoculation in the shape of the corresponding bacterial name initials, EC for E. coli and BS for B. subtilis, the plates were incubated for 24 hours at 37°C. Iodine, which changes color from a yellow-brown to blue-black in the presence of starch, was applied to the agar surface and allowed to stand for 10 minutes .  The E. coli starch agar plate turned completely blue-black which indicated that all the starch was still present (Fig. 2.). This is a negative reaction for the starch hydrolysis test.   The B. subtilis produced a clear zone around the growth which is a positive reaction (Fig. 1.) and indicates that the starch has been removed in the area around the bacterial inoculum   .  B. subtilis produced the enzyme amylase which hydrolyzed starch in the agar.   If the species produces and releases amylase, starch hydrolysis in the agar should occur.

Source

References

1.       Bird, R., and R. H. Hopkins. 1954.  The action of some alpha-amylases on amylase.  Biochem. J.  56 :86–99.
2.      Priest, F. G. 1977. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol. Rev.  41 (3) : 711–753.

Test procedure

  1. 1. Use a sterile swab or a sterile loop to pick a few colonies from your pure culture plate. Streak a starch plate in the form of a line across the width of the plate. Several cultures can be tested on a single agar plate, each represented by a line or the plate may be divided into four quadrants (pie plate) for this purpose.
  2. Incubate plate at 37 °C for 48 hours.
  3. Add 2-3 drops of 10% iodine solution directly onto the edge of colonies. Wait 10- 15 minutes and record the results.

2

Interpretation:

  • Positive test (“+”): The medium will turn dark. However, areas surrounding isolated colonies where starch has been hydrolyzed by amylase will appear clear.
  • Negative test (“-“): The medium will be colored dark, right up to the edge of isolated colonies.

Figure: Two species are inoculated onto a starch plate and incubated at 30°C until growth is seen (plate on the left). The petri dish is then flooded with an iodine solution and photograph taken after 10 minutes (plate on right). Amylase positive species shows a clearing halo around the growth (top line of growth). Amylase negative species does not have this clear halo (bottom line of growth).

Source

Starch Hydrolysis Procedure

Lab One

  1. Using a marking pen, divide the starch agar plate into three equal sectors. Be sure to mark on the bottom of the plate.
  2. Label the plate with the organisms’ names, your name, and the date.
  3. Spot inoculate two sectors with the test organisms.
  4. Invert the plate and incubate it aerobically at 35°C for 48 hours.

Lab Two

  1. Remove the plate from the incubator and note the location and appearance of the growth before adding the iodine. (Growth that is thinning at the edge may give the appearance of clearing in the agar after iodine is added to the plate.)
  2. Cover the growth and surrounding areas with Gram iodine. Immediately examine the areas surrounding the growth for clearing. (Usually the growth on the agar prevents contact between the starch and iodine so no color reaction takes place at that point. Beginning students sometimes look at this lack of color change and incorrectly judge it as a positive result. Therefore, when examining the agar for clearing, look for a halo around the growth, not at the growth itself.)
  3. Record your results in the table provided.

PURPOSE

This test is used to differentiate bacteria based on their ability to hydrolyze starch with the enzyme a-amylase or oligo-l,6-glucosidase. It aids in the differentiation of species from the genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and members of Enterococcus.

PRINCIPLE

Starch is a polysaccharide made up of a-D-glucose subunits. It exists as a mixture of two forms, linear (amylose) and branched (amylopectin), with the branched configuration being the predominant form. The a-D-glucose molecules in both amylose and amylopectin are bonded by 1,4-a-glycosidic (acetal) linkages (Figure 6-83). The two forms differ in that the amylopectin contains polysaccharide side chains connected to approximately every 30th glucose in the main chain. These side chains are identical to the main chain except that the number 1 carbon of the first glucose in the side chain is bonded to carbon number 6 of the main chain glucose. The bond is, therefore, a 1,6-a-glycosidic linkage.

Starch is too large to pass through the bacterial cell membrane. Therefore, to be of metabolic value to the bacteria it must first be split into smaller fragments or individual glucose molecules. Organisms that produce and secrete the extracellular enzymes a-amylase and oligo-l,6-glucosidase are able to hydrolyze starch by breaking the glycosidic linkages between the sugar subunits. Although there usually are intermediate steps and additional enzymes utilized, the overall reaction is the complete hydrolysis of the polysaccharide to its individual a-glucose subunits (Figure 6-83).

Figure 6-83

Starch agar is a simple plated medium of beef extract, soluble starch and agar. When organisms that produce a-amylase and oligo-Le-glucosidase are grown on starch agar theyhydrolyzethe starch in the medium surrounding the bacterial growth. Because both the starch and its sugar subunits are soluble (Clear) in the medium, the reagent iodine is used to detect the presence or absence of starch in the vicinity around the bacterial growth. Iodine reacts with starch and produces a blue or dark brown color; therefore, any microbial starch hydrolysis will be revealed as a clear zone surrounding the growth (Figure 6-84).

Figure 6-84

 

Source

Result interpretation of Starch Hydrolysis Test

Positive test: a clear zone around the line of growth after addition of iodine solution.

Negative test: dark blue colouration of the medium

Questions and Answers on Starch Hydrolysis Test

  • What is Starch

A large carbohydrate molecule with hundred or thousands of glucose subunits. Since starch is so big, bacteria can’t use the valuable glucose molecules in it without first breaking it down.

  • What is the enzyme used in Starch Hydrolysis?

Amylase, which breaks (hydrolyzes) some of the bonds between glucose subunits. Which helps bacteria break down starch.

  • What is the medium used in the Starch Hydrolysis Test?

A Starch Agar plate, which is a standard agar plate with added starch. The starch is clear because it completely dissolves in water.

  • How can you see starch on the Starch Agar Plate?

You add Iodine to the plate after incubation. The Iodine forms a dark blue/black complex with starch but remains its original amber color in the absence of starch.

  • What does a positive Starch Hydrolysis test look like?

After incubation and the iodine is added the iodine will remain its original amber color around the bacteria that has the enzyme Amylase.

  • What does a negative Starch Hydrolysis test look like?

After incubation and the iodine is added the iodine will turn a dark blue/black complex with the starch that is intact.

  • What does a positive Starch Hydrolysis test mean?

It means that the bacteria has the enzyme amylase.

  • What does a negative Starch Hydrolysis test mean?

It means that the bacteria doesn’t have the enzyme amylase.

  • What are the steps of a Starch Hydrolysis Test?
  1. Use a loop to streak each organism in a single line on the surface of the Starch Agar Plate.
  2. Incubate the plate at 37C till next class
  3. Obtain the incubated plates
  4. Coat the surface of each agar with Gram’s Iodine
  5. Observe and record
  • What is the reagent used?

Grams Iodine

  • What is the purpose of the test?

The purpose is to see if the microbe can use starch, a complex carbohydrate made from glucose, as a source of carbon and energy for growth. Use of starch is accomplished by an enzyme called alpha-amylase.

  • How is alpha-amylase activity determined?

A medium containing starch is used. After inoculation and overnight incubation, iodine reagent is added to detect the presence of starch. Iodine reagent complexes with starch to form a blue-black color in the culture medium. Clear halos surrounding colonies is indicative of their ability to digest the starch in the medium due to the presence of alpha-amylase.

  • What medium is used?

The medium used is starch agar. The medium is a nutrient agar to which starch is added.

  • How is the test performed?

An inoculum from a pure culture is streaked on a sterile plate of starch agar The inoculated plate is incubated at 35-37 C for 24 hours. Iodine reagent is then added to flood the growth. Presence of clear halos surrounding colonies is positive for their ability to digest the starch and thus indicates presence of alpha-amylase.

  • What reagents are added?

Iodine reagent is added after incubation to flood the surface of the plate.

REFERENCES

Collins, C. H., Patricia M. Lyne, J. M. Grange. 1995. Page 117 in Collins and Liyne’s Microbiological Methods, 7th Ed. Butterworth-Heinemann, UK. DIFCO Laboratories. 1984. Page 879 in

DIFCO Manual, 10th Ed., DIFCO Laboratories, Detroit, MI.

Lanyi, B. 1987. Page 55 in Methods in Microbiology, Vol. 19, edited by R. R. Colwell and R. Grigorova, Academic Press Inc., New York, NY.

MacFaddin, Jean F. 2000. Page 412 in Biochemical Tests for Identification of Medical Bacteria, 2nd Ed. Lippincott Williams & Wilkins, Philadelphia, PA.

Smibert, Robert M. and Noel R. Krieg. 1994. Page 630 in Methods for General and Molecular Bacteriology, edited by Philipp Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg,  American Society for Microbiology, Washington, DC.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC242086/pdf/aem00178-0237.pdf

https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU1854.pdf

https://jb.asm.org/content/jb/83/2/264.full.pdf

Biochemical Tests: Gram Positive and gram Negative Bacteria

Tests used to identify Gram Positive Bacteria

Mannitol Salt Agar (MSA)

This type of medium is both selective and differential. The MSA will select for organisms such as Staphylococcus species which can live in areas of high salt concentration (plate on the left in the picture below). This is in contrast to Streptococcus species, whose growth is selected against by this high salt agar (plate on the right in the picture below).

The differential ingredient in MSA is the sugar mannitol. Organisms capable of using mannitol as a food source will produce acidic byproducts of fermentation that will lower the pH of the media. The acidity of the media will cause the pH indicator, phenol red, to turn yellow. Staphylococcus aureus is capable of fermenting mannitol (left side of left plate) while Staphylococcus epidermidis is not (right side of left plate).

Glucose broth with Durham tubes

This is a differential medium. It tests an organism’s ability to ferment the sugar glucose as well as its ability to convert the end product of glycolysis, pyruvic acid into gaseous byproducts. This is a test commonly used when trying to identify Gram-negative enteric bacteria, all of which are glucose fermenters but only some of which produce gas.

Like MSA, this medium also contains the pH indicator, phenol red. If an organism is capable of fermenting the sugar glucose, then acidic byproducts are formed and the pH indicator turns yellow. Escherichia coli is capable of fermenting glucose as are Proteus mirabilis (far right) and Shigella dysenteriae (far left).  Pseudomonas aeruginosa (center) is a nonfermenter.

The end product of glycolysis is pyruvate. Organisms that are capable of converting pyruvate to formic acid and formic acid to H2 (g) and CO2 (g), via the action of the enzyme formic hydrogen lyase, emit gas. This gas is trapped in the Durham tube and appears as a bubble at the top of the tube. Escherichia coli and Proteus mirabilis (far right) are both gas producers. Notice that Shigella dysenteriae (far left) ferments glucose but does not produce gas.

*Note – broth tubes can be made containing sugars other than glucose (e.g. lactose and mannitol).  Because the same pH indicator (phenol red) is also used in these fermentation tubes, the same results are considered positive  (e.g. a lactose broth tube that turns yellow after incubation has been inoculated with an organism that can ferment lactose).

Blood Agar Plates (BAP)

This is a differential medium. It is a rich, complex medium that contains 5% sheep red blood cells. BAP tests the ability of an organism to produce hemolysins, enzymes that damage/lyse red blood cells (erythrocytes). The degree of hemolysis by these hemolysins is helpful in differentiating members of the genera Staphylococcus, Streptococcus and Enterococcus.

  • Beta-hemolysis is complete hemolysis. It is characterized by a clear (transparent) zone surrounding the colonies. Staphylococcus aureus, Streptococcus pyogenes and Streptococcus agalactiaeare b-hemolytic (the picture on the left below shows the beta-hemolysis of S. pyogenes).
  • Partial hemolysis is termed alpha-hemolysis. Colonies typically are surrounded by a green, opaque zone. Streptococcus pneumoniae and Streptococcus mitis are a-hemolytic (the picture on the right below shows the a-hemolysis of S. mitis).
  • If no hemolysis occurs, this is termed gamma-hemolysis. There are no notable zones around the colonies. Staphylococcus epidermidis is gamma-hemolytic.

What type of hemolysis is seen on each one of the following plates?

 

Streak-stab technique

Often when inoculating a BAP to observe hemoloysis patterns, investigators will also stab several times through the agar using an inoculating loop. This stab allows for the detection of streptolysin O, a specific hemolysin produced by Streptococcus pyogenes. This hemolysin is inactivated by O2 and is only seen subsurface (in an anaerobic environment) around the stab mark. Note the oval-shaped areas of clearing around the stab marks in the picture below; these are caused by streptolysin O.

Bile Esculin Agar

This is a medium that is both selective and differential. It tests the ability of organisms to hydrolyze esculin in the presence of bile. It is commonly used to identify members of the genus Enterococcus (E faecalis and E. faecium).

The first selective ingredient in this agar is bile, which inhibits the growth of Gram-positives other than enterococci and some streptococci species. The second selective ingredient is sodium azide. This chemical inhibits the growth of Gram-negatives.

The differential ingredient is esculin. If an organism can hydrolyze esculin in the presence of bile, the product esculetin is formed. Esculetin reacts with ferric citrate (in the medium), forming a phenolic iron complex which turns the entire slant dark brown to black. The tube on the far right was inoculated with E. faecalis (positive). The tube in the center was inoculated with a bilie esculin negative organism and the tube on the left was uninoculated.

Sulfur Indole Motility Media (SIM)

This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae.

Sulfur can be reduced to H2S (hydrogen sulfide) either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for H2S production. The organism pictured on the far left is positive for hydrogen sulfide production.

Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole. Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color (indole +). Escherichia coli is indole positive. The organism pictured second from left is E. coli and is indole positive.

SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile than the growth will radiate from the stab mark and make the entire tube appear turbid. Pseudomonas aeruginosa and the strain of Proteus mirabilis that we work with are motile.

Kliger’s Iron Agar (KIA)

This is a differential medium. It tests for organisms’ abilities to ferment glucose and lactose to acid and acid plus gas end products. It also allows for identification of sulfur reducers. This media is commonly used to separate lactose fermenting members of the family Enterobacteriaceae (e.g. Escherichia coli) from members that do not ferment lactose, like Shigella dysenteriae. These lactose nonfermenting enterics generally tend to be the more serious pathogens of the the gastrointestinal tract.

The first differential ingredient, glucose, is in very short supply. Organisms capable of fermenting this sugar will use it up within the first few hours of incubation. Glucose fermentation will create acidic byproducts that will turn the phenol red indicator in the media yelllow. Thus, after the first few hours of incubation, the tube will be entirely yellow. At this point, when the glucose has been all used up, the organism must choose another food source. If the organism can ferment lactose, this is the sugar it will choose. Lactose fermentation will continue to produce acidic byproducts and the media will remain yellow (picture on the far left below). If gas is produced as a result of glucose or lactose fermentation, then fissures will appear in the agar or the agar will be lifted off the bottom of the tube.

If an organism cannot use lactose as a food source it will be forced to use the amino acids / proteins in the media. The deamination of the amino acids creates NH3, a weak base, which causes the medium to become alkaline. The alkaline pH causes the phenol red indicator to begin to turn red. Since the incubation time is short (18-24 h), only the slant has a chance to turn red and not the entire tube. Thus an organism that can ferment glucose but not lactose, will produce a red slant and a yellow butt in a KIA tube (second from the left below). These organisms are the more serious pathogens of the GIT such as Shigella dysenteriae.

If an organism is capable of using neither glucose nor lactose, the organism will use solely amino acids / proteins. The slant of the tube will be red and the color of the butt will remain unchanged (picture on the far right below). Pseudomonas aeruginosa is an example of a nonfermenter.

KIA tubes are also capable of detecting the production of H2S. It is seen as a black precipitate (second picture from the right). Sometimes the black precipitate obscures the butt of the tube. In such cases, the organisms should be considered positive for glucose fermentation (yellow butt). Proteus mirabilis (pictured here, second from right) is a glucose positive, lactose negative, sulfur reducing enteric.

Nitrate Broth

This is a differential medium. It is used to determine if an organism is capable of reducing nitrate (NO3-) to nitrite (NO2-) or other nitrogenous compounds via the action of the enzyme nitratase (also called nitrate reductase). This test is important in the identification of both Gram-positive and Gram-negative species.

After incubation, these tubes are first inspected for the presence of gas in the Durham tube. In the case of nonfermenters, this is indicative of reduction of nitrate to nitrogen gas. However, in many cases gas is produced by fermentation and further testing is necessary to determine if reduction of nitrate has occurred. This further testing includes the addition of sulfanilic acid (often called nitrate I) and dimethyl-alpha-napthalamine (nitrate II). If nitrite is present in the media, then it will react with nitrate I and nitrate II to form a red compound. This is considered a positive result.

If no red color forms upon addition of nitrate I and II, this indicates that either the NO3- has not been converted to NO2- (a negative result), or that NO3- was converted to NO2- and then immediately reduced to some other, undetectable form of nitrogen (also a positive result). In order to determine which of the preceding is the case, elemental zinc is added to the broth. Zinc will convert any remaining NO3- to NO2- thus allowing nitrate I and nitrate II to react with the NO2- and form the red pigment (a verified negative result). If no color change occurs upon addition of zinc then this means that the NO3- was converted to NO2- and then was converted to some other undetectable form of nitrogen (a positive result).

If the nitrate broth turns red (tubes pictured in the center) after nitrate I and nitrate II are added, this color indicates a positive result. If instead, the tube turns red (tube pictured on the left) after the addition of Zn, this indicates a negative result. If there is no color change in the tube after the addition of nitrate I and nitrate II, the result is uncertain. If the tube is colorless (picture on the right) after the addition of Zn this indicates a positive test.

Catalase Test

This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas.

The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. The sample on the right below is catalase positive. The Staphylococcus spp. and the Micrococcus spp. arecatalase positive. The Streptococcus and Enterococcus spp. are catalase negative.

Tests used to identify Gram Negative Bacteria

Oxidase Test

This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain). It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae.

Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple. This is considered a positive result. In the picture below the organism on the right (Pseudomonas aeruginosa) is oxidase positive.

Coagulase test

Coagulase is an enzyme that clots blood plasma. This test is performed on Gram-positive, catalase positive species to identify the coagulase positive Staphylococcus aureus. Coagulase is a virulence factor of S. aureus. The formation of clot around an infection caused by this bacteria likely protects it from phagocytosis. This test differentiates Staphylococcus aureus from other coagulase negative Staphylococcus species.

Taxos A (bacitracin sensitivity testing)

This is a differential test used to distinguish between organisms sensitive to the antibiotic bacitracin and those not. Bacitracin is a peptide antibiotic produced by Bacillus subtilis. It inhibits cell wall synthesis and disrupts the cell membrane. This test is commonly used to distinguish between the b-hemolytic streptococci: Streptococcus agalactiae (bacitracin resistant) and Streptococcus pyogenes(bacitracin sensitive). The plate below was streaked with Streptococcus pyogenes; notice the large zone of inhibition surrounding the disk.

Taxos P (optochin sensitivity testing)

This is a differential test used to distinguish between organisms sensitive to the antibiotic optochin and those not. This test is used to distinguish Streptococcus pneumoniae (optochin sensitive (pictured on the right below)) from other a-hemolytic streptococci (optochin resistant (Streptococcus mitis is pictured on the left below)).

MacConkey agar

This medium is both selective and differential. The selective ingredients are the bile salts and the dye, crystal violet which inhibit the growth of Gram-positive bacteria. The differential ingredient is lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color. Thus organisms capable of lactose fermentation such as Escherichia coli, form bright pinky-red colonies (plate pictured on the left here). MacConkey agar is commonly used to differentiate between the Enterobacteriaceae.

Organism on left is positive for lactose fermentation and that on the right is negative.

Simmon’s Citrate Agar

This is a defined medium used to determine if an organism can use citrate as its sole carbon source. It is often used to differentiate between members of Enterobacteriaceae. In organisms capable of utilizing citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2. If CO2 is produced, it reacts with components of the medium to produce an alkaline compound (e.g. Na2CO3). The alkaline pH turns the pH indicator (bromthymol blue) from green to blue. This is a positive result (the tube on the right is citrate positive). Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.

Spirit Blue agar

This agar is used to identify organisms that are capable of producing the enzyme lipase. This enzyme is secreted and hydrolyzes triglycerides to glycerol and three long chain fatty acids. These compounds are small enough to pass through the bacterial cell wall. Glycerol can be converted into a glycolysis intermediate. The fatty acids can be catabolized and their fragments can eventually enter the Kreb’s cycle. Spirit blue agar contains an emulsion of olive oil and spirit blue dye. Bacteria that produce lipase will hydrolyze the olive oil and produce a halo around the bacterial growth. The Gram-positive rod, Bacillus subtilis is lipase positive (pictured on the right) The plate pictured on the left is lipase negative.

Starch hydrolysis test

This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus. Because of the large size of amylose and amylopectin molecules, these organisms can not pass through the bacterial cell wall. In order to use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-glucosidase into the extracellular space. These enzymes break the starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth. Bacillus subtilis is positive for starch hydrolysis (pictured below on the left). The organism shown on the right is negative for starch hydrolysis.

Methyl Red / Voges-Proskauer (MR/VP)

This test is used to determine which fermentation pathway is used to utilize glucose. In the mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids). The stable production of enough acid to overcome the phosphate buffer will result in a pH of below 4.4. If the pH indicator (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear (first picture, tube on the left).

If the MR turns yellow, the pH is above 6.0 and the mixed acid fermentation pathway has not been utilized (first picture, tube on the right). The 2,3 butanediol fermentation pathway will ferment glucose and produce a 2,3 butanediol end product instead of organic acids. In order to test this pathway, an aliquot of the MR/VP culture is removed and a-naphthol and KOH are added. They are shaken together vigorously and set aside for about one hour until the results can be read.

The Voges-Proskauer test detects the presence of acetoin, a precursor of 2,3 butanediol. If the culture is positive for acetoin, it will turn “brownish-red to pink” (tube on the left in the second picture). If the culture is negative for acetoin, it will turn “brownish-green to yellow” (tube on the left in the second picture). Note: A culture will usually only be positive for one pathway: either MR+ or VP+. Escherichia coli is MR+ and VP-. In contrast, Enterobacter aerogenes and Klebsiella pneumoniae are MR- and VP+. Pseudomonas aeruginosa is a glucose nonfermenter and is thus MR- and VP-.

CAMP Test

CAMP factor is a diffusible, heat-stable protein produced by group B streptococci. This is a synergistic test between Staphylococcus aureus and Streptococcus agalactiae. S. agalactiae produces CAMP factor. S. aureus produces sphingomyelin C, which binds to red blood cell membranes. The two bacteria are streaked at 90o angles of one another. They do NOT touch. The CAMP factor produced by S. agalactiae enhances the beta-hemolysis of S. aureus by binding to already damaged red blood cells. As a result, an arrow of beta-hemolysis is produced between the two streaks. The test is presumptive for S. agalactiae that produces CAMP factor.

In the picture here, Streptococcus agalactiae was streaked throughout the top region of the plate and brought down toward the center of the plate. Staphylococcus aureus was streaked in a straight line across the center of the plate. Rings of hemolysis are evident all around S. aureus, however the hemolysis if greatly enhanced (in an arrow shape) where the S. agalactiae crosses the hemolysis rings.

Urease test

This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus mirabilis is a rapid hydrolyzer of urea (center tube pictured here). The tube on the far right was inoculated with a urease negative organism and the tube on the far left was uninoculated.

Motility agar

is a differential medium used to determine whether an organism is equipped with flagella and thus capable of swimming away from a stab mark. The results of motility agar are often difficult to interpret. Generally, if the entire tube is turbid, this indicates that the bacteria have moved away from the stab mark (are motile). The organisms in the two tubes pictured on the right are motile. If, however, the stab mark is clearly visible and the rest of the tube is not turbid, the organism is likely nonmotile (tube pictured on the left).

Bacterial Staining Techniques I

  1. Complete Lab 1:

Collect your plates from the trays on the side bench. Observe the TSA plates for colonies of various sizes, shapes and colors. Each bacterial or fungal species gives a characteristic colony color and morphology. Draw the colonies observed on both TSA plates in the spaces provided in the Results section of Lab #1. Pick three colonies from either of the TSA plates and describe the colony color and morphology. Also observe the cloudiness (turbidity) of your nutrient broth tube and estimate the number of bacteria per mL (see turbidity table below).

TERMS AND DEFINITIONS

Colony: a single cell divides exponentially forming a small, visible collection of cells. Colonies are observed when bacteria are grown on a solid medium. Each colony usually contains 107-108 bacteria.

Colony morphology: Characteristics of a colony such as shape, edge, elevation, color and texture.

Turbidity: cloudy appearance of a liquid medium due to the presence of bacteria. You can “estimate” the number of bacteria per mL by using the table below.

Turbidity # Bacteria per mL none 0 – 106

light 107

moderate 108

*heavy 109

*Usually bacterial populations do not exceed 3 x 109 bacteria/mL when grown in liquid media.

  1. STAINING MICROORGANISMS
    1. Smear preparation Simple Stains:
    2. Direct stain
    3. Negative stain

TERMS AND DEFINITIONS

Chromophores: Groups with conjugated double bonds that give the dye its color.

Direct, cationic, basic or positive dyes: contain positively charged groups. Examples include methylene blue, basic fuchsin, and crystal violet. These dyes directly bind to and stain the negatively charged surface of bacterial cells.

Negative, anionic, or acidic dyes: contain functional groups that have a negative charge. Examples include eosin, nigrosin and Congo red. These dyes are repelled by the negatively charged surface of bacterial cells. Thus, they stain the background, leaving the bacterial cells clear and bright against a dark background.

Heat Fixation: application of heat to a bacterial smear preparation. This procedure simultaneously kills and attaches the bacteria to the slide.

MICROBIOLOGIST’S TOOLS

 

  1. Smear Preparation

The first step in most bacterial staining procedures is the preparation of a smear. In a smear preparation, cells from a culture are spread in a thin film over a small area of a microscope slide, dried, and then fixed to the slide by heating or other chemical fixatives. A good smear preparation should be…

    1. A thin layer of cells so that individual cells can be observed.
    2. Fixed appropriately to allow repeated washings during staining.

PROCEDURE: (EACH STUDENT)

Note: A good smear preparation is the key to a high-quality stain. Care taken when creating a smear will allow for accurate observations.

  1. Use a slide from your slide box. If necessary, clean the slide using soap and water. Dry the slide using a KimWipe. Place the frosted side of the slide facing up and draw a circle (about the size of a nickel) on the bottom (unfrosted) side of the slide. Place 2-3 loopfuls of water on the slide. Don’t forget to draw a focus line on the top of the slide.
  2. Flame an inoculating needle and allow it to cool. Pick up a “tiny” amount of an Escherichia coli colony and mix it into the drop of water on the slide.

3.Flame the needle and transfer a small amount of a Saccharomyces cerevisiae colony in the same manner to the SAME drop.

You will now have a mixture of E. coli (bacteria, procaryotic cell) and S. cerevisiae (yeast, eucaryotic cell) in the same smear preparation.

  1. Air-dry the slide completely. Heat fix the slide by passing it over the flame 3 times. The slide should be uncomfortable to the skin but not painful. The slide is now ready to be stained as described below.

 S. cerevisiae E. coli

Frosted end facing up

Focus line

 Circle drawn on bottom of slide

  1. Direct stain

The cell wall of most bacteria has an overall net negative charge and thus can be stained directly with a single basic (positively charged) stain or dye. This type of stain allows us to observe the shape, size and arrangement of bacteria.

PROCEDURE: (EACH STUDENT)

    1. Use the smear prepared in the previous procedure. Staining is done at the sink.
    2. Add several drops of Methylene blue, enough to cover the smear, and wait 1 min.
    3. Rinse the slide with water from the squirt bottle and blot the slide with bibulous paper.
    4. Redraw the focus line on the top of the slide if necessary.
    5. Focus on the line with the 10X objective – refer to the microscope focusing procedure described in lab 1. Once you have focused on the specimen using the 10X objective, move the 40X objective lens into position. Use the fine adjustment knob to bring the specimen into focus. Now use the following procedure to view the specimen using the 100X (oil-immersion lens):
      1. Rotate the nosepiece to the empty slot between the 40X and 100X objectives.
      2. Add a drop of oil to slide where the light passes through. The oil has the same refractive index as the glass slide and thus prevents light loss.
      3. Move the 100X objective lens into position. The lens will be immersed in oil.
      4. The specimen will be out of focus but you will probably see a blurry image. Focus using the Fine Focus knob. Turn slowly 1/2 a turn toward you. If the specimen does not come into focus turn back a 1/2 turn to the approximate starting position and then turn a 1/2 turn away from you. If specimen is still not in focus call your instructor over to help you. Never turn the Fine Focus knob more than 1/2 a turn in either direction. NEVER use the Coarse Focus knob when using the 100X objective!
      5. Once the specimen is in focus, find a field that has isolated organisms. Then while viewing the organisms fine-tune the image by gently adjusting the condenser diaphragm to give the best light and adjusting the fine focus to give the sharpest image. If you have difficulty in bringing the image into view, move the stage adjustment back and forth while focusing.

f.After examining the slide – move the oil immersion objective away from the slide. Clean the objective thoroughly with lens paper (NOT KimWipes!) and lens cleaningsolution.

    1. Draw the organisms observed in the microscopic field – record in Results Lab 2.

Note: Saccharomyces cerevisiae is a species of yeast. It is a relatively large single-celled eucaryotic organism. Escherichia coli is a “tiny” rod shaped bacteria (procaryotic).

  1. Negative Stain

In contrast to direct stains that bind to bacteria directly, a negative stain colors the background of a smear rather than the bacteria. These stains have negatively charged functional groups so they will not bind directly to negatively charged bacteria. The advantages of negative staining are: 1) bacteria are not heat fixed so they don’t shrink, and 2) some bacterial species resist basic stains (Mycobacterium) and one way they can be visualized is with the negative stain. However, negative staining does not differentiate bacteria; one can only determine morphology.

PROCEDURE: (EACH STUDENT)

    1. Using a flamed inoculating loop, place 2-3 loopfuls of Congo Red in two separate circles on a clean slide. There is no need to add water to the Congo Red.

 Congo Red

B. subtilis tooth scraping

    1. Using a flamed inoculating NEEDLE, pick up a small amount of Bacillus subtilis and stir it into one drop of Congo Red.
    1. Use a toothpick to scrape material from your teeth near the gumline and stir this into the second drop of Congo Red. Be sure to keep the two drops separate.
    1. Air dry – DO NOT HEAT FIX.
    1. Flood the slide with acid-alcohol (95% ethanol, 3% HCl) until it turns blue. This generally takes ~ 2 seconds. Drain the excess acid-alcohol into the appropriately labeled waste container but do not wash the slide.
    1. Allow the slide to air dry; do not blot.
    1. Examine both smears. First focus using the 10X objective. You will not be able to see individual organisms, but you should be able to focus on the stain. Then move to 40X and finally to the oil immersion lens with oil.

Note: Organisms appear white (colorless) against a blue stained background. Draw a typical microscopic field for each slide in the Results section of this lab.

III. Morphological Unknown

The staining procedures introduced in Labs 2-4 are commonly used by microbiologists to help characterize and identify bacteria. These stains often make it possible to determine the group of organisms to which an unknown isolate belongs. With few exceptions, staining is the first step in identifying a bacterial unknown. Although staining alone does not give sufficient information about the organism to make a definitive identification, it will give some important clues. You will be given an unknown pure culture on which you will perform the various stains as you go through labs 2-4.

PROCEDURE: (EACH STUDENT)

  1. Collect an unknown from the side bench. Record the number of your unknown in the Results section. Your T.A. will also record the number of your unknown. It is important that the same unknown number is used throughout the identification process.
  1. Perform a direct stain (methylene blue) on your unknown. Determine the shape of your unknown and any distinctive arrangements of the cells. Record your observations in the results section following Lab 4.

Ubiquity of microorganisms

  1. UBIQUITY OF MICROORGANISMS

Microorganisms are ubiquitous; that is, they are present nearly everywhere. In this lab you will try to isolate bacteria and other microorganisms from various sources using different types of media.

TERMS AND DEFINITIONS

Culture media (medium, singular): solution of nutrients required for the growth of bacteria.

Agar: a carbohydrate derived from seaweed used to solidify a liquid medium.

Tryptic Soy Agar (TSA): a rich solid medium containing a digest of casein (the principal milk protein) and soy products. It is an all-purpose medium that supports the growth of many diverse organisms.

Tryptic Soy Broth (TSB): a rich liquid medium containing a digest of casein and soy products. It is a general-purpose medium that supports the growth of organisms that are not exacting in their food requirements.

Colony: a visible population of microorganisms originating from a single parent cell and growing on a solid medium.

PROCEDURE: (EACH STUDENT) Collect 2 TSA plates and 1 TSB tube from the side bench.

    1. Moisten a sterile swab with sterile water (see bottle on your bench). Using this swab, collect a sample from any surface or object (e.g. doorknobs, shoes, drinking fountain, a strand of hair, various body parts, etc.). Try whatever interests you and be creative.
    2. After the sample has been collected, inoculate a Tryptic Soy Agar (TSA) plate by gently rolling the swab over the surface of the agar. Discard the used swab into a biohazard container.
    3. Label the bottom of the plate with your name, lab section #, date, and the source of the sample. Write on the outer edge so that the markings won’t interfere with observing the colonies growing on the plate.
    4. Inoculate a second Tryptic Soy Agar plate by the following procedure. Open the lid of the plate, place it close to your mouth and cough hard 3 times onto the plate. Place the lid back on. Correctly label your plate.
    5. Inoculate a tube of Tryptic Soy Broth by removing the cap, putting your thumb over the top of the tube, and inverting the tube several times. Replace the cap. Label the tube with your name and lab section using a piece of tape. Do not write directly on the cap or tube.
    6. Incubation: After inoculating culture media with microorganisms, it is usually incubated at a temperature that most closely mimics the organisms’ natural environments.

*a. TSA swabbed plate – room temperature (RT)

*b. TSA cough plate – body temperature–37˚C incubator

c. TSB tube – RT

*Plates are always incubated in an inverted position (agar side up). There are only a few exceptions to this rule that you will see later in the course.

II. MICROSCOPY

In this exercise you will become familiar with a bright field microscope that you’ll be using throughout the semester.

TERMS AND DEFINITIONS

Microscope: a device for magnifying objects that are too small to be seen with the naked eye.

  1. Simple microscope: single lens magnifier
  2. Compound microscope: employs two or more lenses

Parfocal: the objective lenses are mounted on the microscope so that they can be interchanged without having to appreciably vary the focus.

Resolving power or resolution: the ability to distinguish objects that are close together. The better the resolving power of the microscope, the closer together two objects can be and still be seen as separate.

Magnification: the process of enlarging the size of an object, as an optical image.

Total magnification: In a compound microscope the total magnification is the product of the objective and ocular lenses (see figure below). The magnification of the ocular lenses on your scope is 10X.

Objective lens ◊ Ocular lens = Total magnification For example:  low power: (10X)(10X) = 100X high dry: (40X)(10X) = 400X

oil immersion: (100X)(10X) = 1000X

Immersion Oil: Clear, finely detailed images are achieved by contrasting the specimen with their medium. Changing the refractive index of the specimens from their medium attains this contrast. The refractive index is a measure of the relative velocity at which light passes through a material. When light rays pass through the two materials (specimen and medium) that have different refractive indices, the rays change direction from a straight path by bending (refracting) at the boundary between the specimen and the medium. Thus, this increases the image’s contrast between the specimen and the medium.

One way to change the refractive index is by staining the specimen. Another is to use immersion oil. While we want light to refract differently between the specimen and the medium, we do not want to lose any light rays, as this would decrease the resolution of the image. By placing immersion oil between the glass slide and the oil immersion lens (100X), the light rays at the highest magnification can be retained. Immersion oil has the same refractive index as glass so the oil becomes part of the optics of the microscope. Without the oil the light rays are refracted as they enter the air between the slide and the lens and the objective lens would have to be increased in diameter in order to capture them. Using oil has the same effect as increasing the objective diameter therefore improving the resolving power of the lens.

MICROSCOPE COMPONENTS

 

MICROSCOPE CARE

    1. Never slide a microscope across a bench surface. Always carry a microscope with both hands. One hand should be placed on the arm and the other should support the base.
    2. Microscopes should be cleaned both before and after use. Use ONLY lens paper and lens cleaner. Kleenex, paper towels and even Kimwipes can scratch the lenses.
    3. ONLY use oil when using the100X oil immersion lens. DO NOT get oil on the other objective lenses.
    4. Store microscopes with the 10X (low power) objective lens in position or such that the region lacking a lens is in position. Turn the light intensity all the way down.
    5. DO NOT wrap the cord around the microscope. Instead, fold the cord and place it between the arm and the stage or beneath the stage.
    6. Use the course adjustment focusing knob to lower the stage towards the light source. DO NOT crank down on the knob!
    7. Replace the dust cover before putting the microscope away.

PROCEDURE: (EACH STUDENT) The primary objective of this exercise is to gain experience in using the bright field microscope. You will observe the microbial life present in pond water and hay infusions as you practice working with the microscope.

  1. Place a slide on the lab bench frosted side up. The frosted section should feel rough.

2.Draw a line on the slide with a Sharpie marker. This line will be used to help youfocus.

Frosted glass (rough side up)

Marker line

  1. Choose to observe either the pond water or hay infusion. Collect some of the chosen sample using a transfer pipette. Be sure to stir it and then pick up some of the “gunk” from the bottom of the jar. Put a drop of the sample next to the line on the slide.
  2. With your forceps, pick up a coverslip and place it on top of the sample. Avoid bubbles by putting the cover slip down at an angle.
  3. View the sample using your microscope:
    1. Lower the microscope stage a little in order to secure the prepared slide onto the stage using the spring-loaded slide holder.
    2. Turn on the main power switch and adjust the light until the Voltmeter reads 2. Be certain that both the field iris diaphragm and the aperture iris diaphragm are open and that the condenser is set to 0.
    3. Position the 10X objective lens directly above the focus line on the slide. Use the coarse adjustment knob to bring the stage as close to the 10X lens as is possible. Now use the fine adjustment knob to back the stage away until the line comes into focus (*Note – If you see more than one circle of light when looking in the oculars, move these lenses until only one circle of light is present).
    4. Move the slide to view the sample. Try to identify some of the organisms. You may move the high dry objective into place to get a better look at the tiny microbes, but please do not use the oil immersion lens (100X) at this point.
  4. Draw some of the organisms you observed in the results section for Lab 1. When you have finished observing the slide, remove it from the mechanical stage and discard the coverslip in the glass disposal container. Wash and save your microscope slide.
  5. Wash the slides under tap water and store them in a slide box provided in your drawer. Do not place the slides back into the original box.
  6. Every student should have 5 slides in a slide box for future use. Using the colored tape on the lab bench, label your box with your name and lab section.
  7. Before you put your microscope away, ALWAYS do the following:
    1. Turn off the power and place the 10X objective lens or region lacking a lens into position. Turn the light intensity all the way down.
    2. Clean each and every lens (objectives and oculars) with LENS PAPER and cleaning solution. Never, never, never use any other kind of tissue or paper towel.
    3. If using oil, clean up any spilled oil present on other parts of the microscope.
    4. Unplug the microscope, fold the electric cord and place it behind the stage–DO NOT wrap cord around the arm of the microscope.
    5. Replace the dust cover on the microscope and carefully put the microscope in the cabinet with the

ARM FACING OUT.

NOTES

LAB 1 RESULTS

  1. UBIQUITY OF MICROORGANISMS
    1. Draw the colonies observed on the TSA plates.

Note: You will be able to make these observations during LAB 2, AFTER the plates have been incubated.

  TSA Swab Plate TSA Cough Plate

    1. In your own words describe the Colony Morphology and Color of 3 different colonies from either of the TSA plates.

Colony 1  

 

Colony 2  

 

Colony 3  

 

    1. Does the nutrient broth tube show growth of bacteria?   Based on the amount of turbidity, estimate the number of bacteria/mL present in the nutrient broth.

Whodunit? The False Accusations Of An Every Day Staphylococcus epidermidis

Daily Amino Reporter

It was just a quiet day in local suburbia when panic struck. Staphylococcus epidermidis, the local fauna, was being accused of accessory to infection. The local authorities are performing many tests to cause questionable doubt, but S. epidennidis’s lawyers are fighting it all the way. “We want to know the right strain is caught in this unfortunate crime.” Lawyer M. Whitblodcelle commented. The particular infection in question: Meningitis. This reported knows for a fact that S. epidermidis is a cause of this infection, but the most violent perpetrator is Staphylococcus aureus.

The question is who the bystander was and who is the guilty party. “We are assuring that all of the proper tests are being performed on my client” Whitblodcelle commented. The test results have been obtained from an unnamed source especially for the Daily Amino. On the night of his arrest S. epidermidis was a semi-opaque color against his auger and was coldly given the number 130. After his one phone call to renowned microbiologist Dr. Ozzy Drix, he was immediately shuffled off to the direct staining room. Officer Methylene blue sat on S. epidermidis for one minute until she got all the information she needed. Results from her test proved that S. epiderrnidis stained blue and had a cocci shape. He also showed signs of staphylo arrangement.

Officer Blue stated “it is too early to tell anything at this point, there are thousands of people in this city with those signs.” After a 500,000,000 protein bail was put on his head S. epidermidis spent the next few nights in the slammer.

The next few days brought test after test. Early, just after a breakfast of organic compounds, S. epidermidis was escorted into one of the most important labs in the police force. It was time for his gram stain. Officer Violet and Officer Safranin presided the testing. After being stained, rinsed and stained again, S. epidermidis finally talked. There was no denying that he was a purple, cocci, staphylo and Gram Positive cell. The stress was taking a toll on the poor cell though because it seemed like a few of his arrangements had slipped into a diplo formation. “I really wished that he wouldn’t have been so quick to admit that he was Gram Positive” S. epidermidis’s girlfriend Bacillus subtilis commented. “Now everyone will know we are opposites.”

Unfortunately, things were going to get more confusing for the couple. In a daring move to test S. epidermidis accused hidden lipids, Special agent Acid-fast was brought in to perform his stain for the evidence locker. S. epidermidis came out nonacid fast and very blue. He did test positive again for staphylo cocci shape. “One of the most consistent shape tester I have ever seen” Acid fast’s assistant Miss Acid Alcohol commented.

The day was long and grueling, but he was not done yet. The Endospore Agency for the Mental Health of All Domains brought in Ms. M. Green came in and gave S. epidermidis a steam bath with herself to see if he could survive this whole ordeal by producing endospores. The test came back negative, that he was not harboring endospores. After this test he was looking a little pink, but still in staphylo cocci formation. Will all these tests prove him of this heinous crime of infection? Stay tuned to the Daily Amino for more answers.

Fight Night:”Kick’m While They’re Down”

 

Brandie Yeik MOLB 2210 – February 10, 2004

The place was packed, the crowd was on their feet. Friday night, at the Immunocompetent arena in Las Virulence, hit record turbidity of 109, which was no surprise to all the sports fans. Described as the fight of the century, Cystic Fibrosis and Pseudomonas aeruginosa battled it out for the Prokaryotic World Boxing championship title. The defending champion, Pseudomonas aeruginosa, looking a pale cream color, entered the ring flaunting the championship belt. P. aeruginosa’s smooth rod shape, and dominating bright blue color showed when coach Direct Stain, prepared and charged him up for the big fight.

A fruity smell filled the arena (Anderson 2004). This was going to be one that would go down in the history books! The opponent, Cystic Fibrosis, was a new and upcoming star. His most impressive fight earlier in the season was his defeat over Exocrine Gland from Lungs, Indiana (Toder 2002). He entered the ring wearing his signature mucous covered shorts.

“LETS GET READY TO RUMIVIMMMBLE!”

In the first few minutes of the first round, P. aeruginosa, was thrown a cheap move by Cystic Fibrosis, and it seemed as if it might be a short fight. Cystic Fibrosis dosed P. aeruginosa with a strong line of antibiotics. Down for what seemed like the final count, P. aeruginosa stood back up and fought till the end of the first round thanks to his trusty capsule that blocked the antibiotics. Back in the corner he was rejuvenated and took to a different tactic for the remainder of the fight. He figured if he could intimidate Cystic Fibrosis maybe he could scare him into loosing the fight. P. aeruginosa would show his true mean side and change into his Gram-negative costume. Totally dyed pink and showing his true cell wall, he took to the center of the ring. This tactic seemed to do its job. Cystic Fibrosis slowly began to weaken. By the end of the second round Cystic Fibrosis was visibly loosing. kV-g.

The third round was the final, and most exciting round. As the two took the center of the ring, Cystic Fibrosis was slow to his feet, and maybe even a little dizzy. It only took one hard upper cut to knock. Cystic Fibrosis to the ground. The crowd went crazy. However, this was not the end of the fight. At least not for P. aeruginosa. As Cystic Fibrosis lay flat on the ground, weak and vulnerable/ P. aeruginosa proceeded to kick and whip the disease with his one flagellum (Van Deiden 1998). The defenseless opponent had no more ability to fight back, and yet the relentless bacteria proceeded to, well, “knock the mucous” out of the defenseless disorder.

This brutal display of fighting continued for many minutes until a brave antibiotic finally took to the ring and ended the slaughter. Comxnents from the two time champion were the following: “I plan to celebrate this victory by going to the local acid-fast staining bar, having a few alcohol washing and see if I can hold my color, I never have been able to hold my color that well, it kinda makes me blue in the face. I have been known as a non-acid fast kinda guy, but after a win like this, I want to celebrate.” It was reported that later in the night he did indeed have a crazy celebration. It was rumored that a. crazed fan poured a full glass of endospore stain on his pants, which, embarrassing for him, revealed he was not wearing any endospores under his cell wall.

References

Anderson, Nester, Pearsall, Roberts. Microbiology: A Human Perspective. McGraw-Hill 2004. p. 250, 61,281-282.

Toby Kenneth. Pseudomonas aeruginosa. University of Wisconsin-Madison Department of Bacteriology. 2002.

Van Delden, Iglewski. Cell-to-Cell Signaling and Pseudomonas aeru2inosa Infections. University of Rochester School of Medicine and Dentistry. 1998.

 

New Bacteria Discovered in Local Hospital

Brian Hardy

Pallet Town-LT-Recently, a large amount of nosocomial infections have been reported at the Pallet Town Hospital. When asked about what may be the cause of these nosocomial infections, Nurse Joy, the Nurse Manager of the center, assured us that they are handling the situation well. “We realized that the patients getting sick were cancer patients,” Nurse Joy told us. “All these patients had indwelling catheters and incidentally they all had urinary tract infections.” The hospital decided that more research needed to be done to determine what was causing these infections.

To accomplish this, they hired a well-known Microbiologist, Professor Oak, to do some investigating. “First, I had to obtain a sample of the bacteria I was trying to identify,” Oak informed us. “We obtained a urine sample from a sick patient and began to research right away.” Oak was able to grow the bacteria in his laboratory. He observed the bacteria and used these observations to identify the microorganism. “When we looked at the bacteria first, we saw that it was white in color and grew randomly in the test tube. We then stained it and looked at its arrangement. We found that the bacteria were spherical, or cocci, and arranged in irregular clusters, like grapes.” Oak informed us that characteristics like these are very important in identifying the bacteria in question.

“Next we performed a Gram stain to determine whether it was Gram negative of Gram positive. After the staining procedure, we discovered that the bacteria were Gram positive. This helped us get a closer idea as to what we were dealing with.” Gram staining is a very important step in identifying bacteria. When we know whether a bacterium is Gram negative of Gram positive, we are able to narrow the search for identification. “We also performed some other tests to help narrow our search. By staining, we determined that it was not Acid-fast and not an endospore former.” The researchers soon discovered that they had found something special.

“The bacteria we isolated had never been seen before. We soon realized that we had discovered a bacterium. We decided to name it Staphylococcus epidermidis. We then had the task of finding out how these bacteria got in patient’s bladders.” The research team took samples from various areas in the hospital and soon discovered that Staphylococcus epidermidis was most commonly found on the skin. This is called normal flora. They used this information to hypothesize that the bacteria found their way into bladders through catheter tubes. The normal flora, which is usually harmless, caused problems to the patients because they had low immune systems and were unable to fight off the infection. Nurse Joy assures us that sterile gloves are now worn when catheters are inserted and other invasive procedures are done. The Hospital is sure that when sterile procedures are used, their high amount of nosocomial infections will decrease. Research for this article came from class notes and Microbiology: A Human Perspective.

Dichotomous Key Microbiology

General Info:

Example 1

Example 2

Example 3

Example 5

Material to accompany labs 1-14

Material to accompany labs 15-28