LAB 6-EXTRACTION OF PLASMID DNA USING GF-1 PLASMID DNA EXTRACTION KIT.

INTRODUCTION

                 DNA is the blueprint of life,if one interested in tuning the properties of life,DNA is the ultimate goal.In bacterial DNA recombinant technology,we often encounter plasmid which is a circular DNA which can replicate independently.Plasmid can grant various desirable characteristics to target bacteria,eg:Antibiotic resistant as shown in MRSA.

                  A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

                 Plasmid is small(hundreds to thousand of Kilobase) compared with the DNA. Plasmid can be transferred interspecific o r intraspecific. Before we can utilized plasmid as vector for transferring gene, we need to purify and isolate the plasmid in order to give a better access to restriction enzyme. Protease is also use to remove protein fragment that will contaminate the sample.
To extract DNA from a mixture of protein ,lipid and carbohydrate,the procedure is crucial in the experiments. Preparation are sort by size, minipreparation are used in the process of molecular cloning to analyze bacterial clones and yield a small weight of DNA.

               DNA filtration is the first and the most important step in DNA recombinant technology as the undesirable effect will amplified as the DNA will replicate many times. Hence, protocol must be followed in order to produce a consistent result in the product.

Objective

To understand the proper method needed to extract DNA from bacteria.

Method:

1.  1ml of bacteria culture is pelleted by centrifugation at 6 000 x g for 2 min at room temperature. The supernatant is decanted completely.

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2.   100µl Buffer R1 is added to the pellet and the cells are resuspended completely by   pipetting up and  down.

3.    For Gram-negative bacteria strains, 10µl lysozyme is added into the cell suspension. For Gram-positive bacteria strains, 20µl lysozyme is added into the cell suspension. The cell suspension is mixed thoroughly and incubated at 37°C for 20 min.

4. The pellet digested cells is centrifuged at 10 000 x g for 3 min. The supernatant is decanted completely.

1    5.  The pellet is resuspended in 180µl of Buffer R2 and 20µl of Proteinase K is added. It is mixed    thoroughly and incubated at 65°C for 20 min with occasional mixing every 5min.

      6.  400µl of Buffer BG is added and mixed thoroughly by inverting tube several times until a homogeneous  solution is obtained. The sample is incubated for 10 min at 65°C. 

       7.  200µl of absolute ethanol is added. The sample is mixed immediately and thoroughly.

       8.  The sample is transferred into a column assembled in a clean collection tube and centrifuged at 10 000 x    g for 1 min. The flow through is discarded.

      9.  The column is washed with 750µl of Wash Bufferand centrifuged at 10 000 x g for 1 min. The flow through is discarded.

    

     10.  The column is centrifuged at 10 000 x g for 1 min to remove residual ethanol.

    

     11.   The column is placed into a clean microcentrifuge tube. 100µl of preheated Elution Buffer is added directly onto column membrane and is stood for 2 min. The column is centrifuged at 10 000 x g for 1 min to elute DNA.

     12.  The sample is placed into the spectrophotometer to measure the reading of absorbance at 260nm and 280nm.

Results

Gram	Type of bacteria	260	280	Ratio (260/280)
Gram positive	Lactobacillus fermentation 8312	0.087	0.044	1.98
	Lactobacillus breris	0.130	0.181	0.72
Gram negative	E.coli	0.095	0.065	1.45
	Salmonella sp	0.109	0.094	1.16

Discussion:

From the calculation of result that we got from the photo spectrometer , the ratio of OD260 to OD280 for  Lactobacillus fermentation was  1.98. The value was very close to 2.0 and it showed that higher purity of RNA and contained less contaminants in the solution. Besides, the ratio of OD260 to OD280 that we got for  Escherichia coli and Salmonella were 1.45 and 1.16 respectively.The values were less than 1.6 and it showed that the solution contained a lot of contaminants. Not only that, the ratio of OD260 to OD280 for Lactobacillus breris was 0.72 and it showed that the solution contained even more contaminants like proteins , phenols and other contaminants.

So, we can know that the best ratio of OD260 to OD280 is 1.6-1.9. If the ratio is less than 1.6, the solution is contained some contaminants. However,if the ratio is more than 1.9,which mean that the solution is contained a higher purity of RNA. If the solution is in the range of such ratio stated, the solution contained a higher purity of DNA.

Other than that, we have discussed that some purposes of using different reagents for this experiment. For example, the purpose of adding lysozyme is to damage the bacterial cell wall by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan. Besides, the purpose of adding proteinase K is to cleave adjacent to carboxyl group of aromatic and aliphatic acid, it also includes in comprising the peptide cross linking bridge of peptidoglycan layer of cell wall of bacteria. The purpose of adding ethanol is to remove the salt out from nucleid acid, so the DNA molecules can become entangled with each other. The purpose f adding buffer R1 is to provide an optimum condition for Hydrogen to function effectively; Adding R2 is to privide an optimum condition for proteinase K to function efficiently; Adding elution buffer is to dilute the mixture of the solution and lastly, adding buffer BG is to trap the nucleid acid.

 Belows are some reasons of getting a low yield of DNA plasmid:

1.  Certain types of protein present in the mixture of protein and DNA.  
2. Plasmid DNA could not digested well/ did not deposit into wall of gel.
3.   Plasmid is denatured or incomplete protein denaturation.
4.  Concentrations of different samples.
5.   Efficiency of buffer elution is low.
6.  Cell suspension is incompleted.
7.   RNA contamination.

Conclusion

As a conclusion, in this experiment, we did not get the ratio as in the required ratio. This is because there are some mistakes that happened during handling out this experiment. So, in order to obtain the best ratio of OD260 and OD280 which are between 1.7 to 1.9, we have to ensure that the samples are in the correct required concentrations. Besides that, we also have to ensure that there is no contamination such as protein in the sample. Otherwise, it will affect the reading of the spectrophotometer and it will cause the ratio obtain are not in the range between 1.7 to 1.9.

LAB 5 DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS 

Introduction.

An antimicrobial is an agent that kills microorganisms or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibacterials (commonly known as antibiotics) are used against bacteria and antifungals are used against fungi. They can also be classed according to their function. Antimicrobials that kill microbes are called microbicidal; those that merely inhibit their growth are called microbiostatic. Disinfectants such as bleach are non-selective antimicrobials.

Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain . They are typically considered to be narrow spectrum antibiotics, though this has been debated.  They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse.

In fermented foods, lactic acid bacteria (LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with industrial potential have been  purified  and  characterized. These bacteriocins have been reported to inhibit the growth of many pathogens. In this experiment, we are going to examine and discuss the effects of LAB strains on Escherichia coli (E.coli)  and Staphylococcus aureus (S.aureus).

Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. There are several large categories of bacteriocin which are only phenomenologically related. These include the bacteriocins from gram-positive bacteria, the colicins, the microcins, and the bacteriocins from Archaea. The bacteriocins from E. coli are called colicins (formerly called 'colicines,' meaning 'coli killers'). They are the longest studied bacteriocins. They are a diverse group of bacteriocins and do not include all the bacteriocins produced by E. coli. For example the bacteriocins produced by Staphylococcus warneri are called as warnerin or warnericin

E. coli is a Gram-negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms).It normally lives inside your intestines, where it helps your body break down and digest the food you eat. Unfortunately, certain strains of E. coli can get from the intestines into the blood. This is a rare illness, but it can cause a very serious infection.

Objective

·         To determine the antimicrobial effects of extracellular extracts of selected LAB strains.

Materials and reagents:

·         MRS broth

·         Sterile filter paper

·         Forceps

·         Sterile universal bottles

·         Cultures of LAB and spoilage/pathogenic organisms

·         Bench-top refrigerated centrifuge.

·         Incubator 30 and 37 degree celcius .

·         UV/V is spectrophotometer.

·         Distilled deionized water

·         Trypticase soy agar

·         Brain heart infusion agar

·         Yeast extract

Part 1 Determination of bacteriocin activity via agar diffusion test

1. All the petri dishes are labelles according to the spoilage organisms and starins of LAB used .
2. One strain of spoilage organism and one strain of LAB will be used in each plate. The plate is divided into 2, each side for one replicate.
3. Each group will have 3 strains of LAB and 3 trains of spoilage/pathogenic organisms.
4. 1. 
5. 10 ml of tryticase soy-yeast extract agar (TSAYE) is loaded into the labeled petri
6. dishes and the agar is ensured to cover the entire surface of the plate. The agar is waited until it became solidified.
7. 2 ml of the broth containing the spoilage organism is inoculated into 10 ml of the brain hear infusion (BHI) agar and then it is being vortex.
8. The mixture is loaded on the top of the TSAYE agar layer and is ensured that it covers the entire surface. The gar is waited until it became solidified. 
9. The broth containing LAB cultures is centrifuged. The supernatant will be used as extracellular extracts
10. A sterile filter paper disk is aseptically picked up with sterile forceps and is dipped into the extracellular extract. The excess extract is ensured to has drained off.
11. The paper disk is placed on the top of the solidified BHI agar.
12. Next, the plates is incubated for 24 – 28 hours at 37˚C.
13. The inhibiton zones is measures in cm and the readings is recorded upon incubation.

Part 2: Determination of bacteria activity via optical density

1. Broth containing LAB culture is centrifuged. The supernatant will be used as extracellular extracts.
2. 3 strains of LAB and 3 strains of spoilage or pathogenic organisms are obtained.
3. 5 ml of double-strength MRS is added with 1 ml of cultures containing spoilage or photogenic bacteria. The mixture is then vortex.
4. A serial dilution of the extracellular extracts is prepared. ( diluted 0x, 2x, 10x, 50x, 100x)
5. 5 ml of each extracellular extracts dilution is added into the mixture as prepared in step (3).
6. The mixture is incubated for 12-15 hours at 37 °c.
7. A control using 5 ml of double-strength MRS, 1 ml of cultures containing spoilage or pathogenic bacteria ,and 5 ml of sterile peptone is prepared. The mixture is incubated for 12-15 hours at 37 °c.
8. A negative control for auto-zero is prepared via the spectrophotometer. 5 ml of double-strength MRS is added with 2 ml of distilled water. (Need not incubate)
9. Upon incubation, the optical density of the spoilage or pathogenic bacteria is measured at 600nm. The same is performed for control as well.
10. One arbitrary unit (AU) is defined as the dilution factor of the extracellular extract that inhibited 50% of the spoilage or pathogenic bacteria growth and expressed as AU/ml.
11. 50% of the spoilage or pathogenic bacteria growth are determined from the OD₆₀₀ of the control.

LAB 4 : SOURCES OF CONTAMINATION AND INFECTION

Introduction:

Microorganisms are very tiny one-celled organisms, viruses, fungi, and bacteria, and are found everywhere in the world. They are found in all living things, plants and animal. There are more microorganisms on and inside your body than there are cells that make up your entire body. Microorganisms can live in the air, on land, and in fresh or salt water environments. Some of them, pathogens, can be harmful and causes diseases, but there are some microorganisms that are needed for living things to survive.

Airborne microbes cause a lot of illnesses and diseases in humans. Microorganisms can enter the air when a human or animal sneezes, or by the wind picking up the light particles and blowing them where humans are. When a human sneezes microorganisms leave the lungs at around 200 miles per hour. Some of the microorganisms that are growing in the mucus in the respiratory tract enter the air with the moisture particles that are sneezed out of the lungs. These microorganisms can be breathed into the lungs of another person and that person could get sick.

Many normal flora provide direct benefits, such as making vitamins or aiding digestion. Even if normal flora microbes merely take up space and resources, they help prevent pathogens (disease causing microbes) from easily invading the body and causing illness. Although there are many different species of normal flora, these bacteria, fungi and protozoans typically fall into one of two categories:

(a)      Resident microbiota

(b)      Transient microbiota

        

   Resident Microbiota

            The body’s resident microbiota are just that — residents. These species are life-long members of the body's normal microbial community, but are not found everywhere. There are many areas of the human body that remain axenic, and, in the absence of disease, are never colonized by normal flora. Axenic areas include the body cavity, lungs, central nervous system, circulatory system and upper urogenital regions.

            Resident microbiota typically colonize the surface of the skin, mucous membranes, digestive tract, upper respiratory system and distal portion of the urogenital system. These microbes have a commensal relationship with their host, meaning that they do not cause harm while they benefit from feeding on the cellular waste and dead cells of the host's body.

  Transient Microbiota

Transient microbes are just passing through. Although they may attempt to colonize the same areas of the body as do resident microbiota, transients are unable to remain in the body for extended periods of time due to:

• competition from resident microbes
• elimination by the body’s immune system
• physical or chemical changes within the body that discourage the growth of transient microbes

Objective:

     To determine the microorganism in the air and from healthy humans.

Material and reagents:

     Molten nutrient agar(commercial and own prepared), sterile water, sterile petri dishes, sterile clinical swab, pipette and tips.

Procedure:

Air:

1.  The molten agar is poured into sterile petri dish and it is cooled.
2. The lid is removed from the plate and it is left resting on the side of the plate, facing down. (The lid of the petri dish is never inverted.) The plates are left exposed for about 5 minutes.
3. The lids are replaced and incubated at 37°C for 48 hours.

Hands:

1. The hand is washed using sterile water. We did not use soap.
2. An automatic pipette is used to transfer 1ml of wash water to the petri dish.
3. The molten nutrient agar is added to the petri dish.
4. The lids of the petri dish are replaced and are gently rotated the dish until the wash water is thoroughly mixed with the molten agar. The agar is not allowed to contact the lid of the dish.
5. After the agar has set, the dish is inverted and incubated at 37°C for 48 hours.

Ear:

1. The molten agar is poured into sterile petri dish and it is cooled.
2. Using extreme care, a sterile swab moistened is rubbed with sterile isotonic solution into the ear of the subject.
3. The swab is used to inoculate the labeled plate. The inoculum is distributed as in the streak method.
4. The dish is inverted and incubated at 37°C for 48 hours.

Normal breathing:

1. The molten agar is poured into sterile petri dish and it is cooled.
2. The lid is removed and the plate is held about 15cm from your mouth. We have breathed normally but directly onto the plate for one minute. The lid is replaced.
3. The dish is inverted and incubated at 37°C for 48 hours.

Violent coughing:

1. The molten agar is poured into sterile petri dish and it is cooled.
2. The lid is removed and the plate is held about 15cm from your mouth. We have coughed violently onto the agar. The lid is replaced.
3. The dish is inverted and incubated at 37°C for 48 hours.

Results:

Air ( commercial )

Air ( own prepared )

Hand ( commercial )

Hand ( own prepared )

Ear ( commercial )

Ear ( own prepared )

Normal breathing ( commercial )

Normal breathing ( own prepared )

Violent coughing ( commercial )

Violent coughing ( own prepared )

Discussion:

This pile of cells originates from one cell and is called a bacterial colony. Each species of bacteria produces a colony that looks different than the colonies produced by other species of bacteria. Examination of the form and structure of bacterial colonies is termed colony morphology and is one of the first steps in characterizing and identifying a bacterial culture.

These are the characteristics used to accurately and consistently describe the morphology of a bacterial colony:

• Size

The size of the colony can be described in two ways. The more accurate technique would be to measure the diameter of the colony with a ruler and report the size in millimeters. The second technique would simply be to describe the colonies as punctiform (tiny pinpoints), small, medium, or large.

• Shape

Shape refers to the overall appearance of the colonies. The descriptors here are punctiform, circular, irregular, filamentous (has individual thin projections), or rhizoid (has thin, branching projections).

• Colour

Some bacteria produce pigments, giving the colony a distinct color. Pigments can span the entire color spectrum. Recording the color is the first step. In addition to describing the color, this is also the time to identify if the colony is opaque (you can't see through it), translucent (you can see through it), dull, or shiny.

• Texture

Texture refers to the characteristics of the colony surface. Colonies can be dry, mucoid (thick, stringy, and wet), moist, smooth, rough, rugose (wrinkled), or contain concentric rings.

• Height

The colony height, or elevation, is a description of how the colony grows vertically. To see the elevation of the colonies, it may be helpful to look through the side of the petri dish. The descriptors here are flat, raised, convex (sloping up from the edges), pulvinate (sloping steeply from the edges and very high in the center), and umbonate (has a raised center).

• Margin

Margin describes the borders of the colony. The edge can be entire (smooth, with no projections), undulate (wavy), lobate (lobed), filamentous, or rhizoid.

1) Morphology of the bacteria colonies which air is the source of contamination

A) COMMERCIAL

-          Form : Filamentous

-          Elevation : Raised, crateriform

-          Color : Cloudy, yellow

-          Margin : Filiform

-          Surface : smooth

B) OWN PREPARED

-          Form : Circular

-          Elevation : Umbonate

-          Color : Cloudy

-          Margin : Entire

-          Surface : Rough

2) Morphology of the bacteria colonies which hand is the source of contamination

)     
        A) COMMERCIAL

-          Form : Rhizoid

-          Elevation : Flat  

        Texture : Moist

-          Color : Cloudy

-          Margin : Entire

-          Surface : Rough

  B) OWN PREPARED

-          Form : Irregular

-          Elevation : Flat

        Texture : Moist

-          Color : Cloudy

-          Margin : Curled

-          Surface : Rough

3) Morphology of the bacteria colonies which ear is the source of contamination

)       A) COMMERCIAL

-          Form : Circular

-          Elevation : Raised  

        Texture : Dry

-          Color : Buff , dull yellow

-          Margin : Entire

-          Surface : Skinny , smooth

  B) OWN PREPARED

-          Form : Circular , Irregular

-          Elevation : Flat ,Raised

        Texture : Dry

-          Color : Cloudy , Opaque

-          Margin : Entire

-          Surface : Skinny , smooth

4) Morphology of the bacteria colonies of which normal breathing is the source of contamination

      A)COMMERCIAL

-      Form: undulate, irregular, circular

Elevation: raised, flat

Colour: opaque, cloudly

Surface: smooth

B) OWN PREPARED

Form: circular

Elevation: raised, flat

Colour: opaque

Margin: entire

Surface: wrinkled, smooth

5) Morphology of the bacteria colonies which violent coughing is the source of contamination

        A) COMMERCIAL

-          Form : Circular , Irregular

-          Elevation : Flat , raised

        Texture : Moist

-          Color : Cloudy

-          Margin : Entire

   B) OWN PREPARED

-          Form : Circular , rhizoid

-          Elevation : Convex , crateriform

-          Color : Cloudy

-          Margin : Undulate

             There are many different compounds that may pollute our air at any given time or place.  Scientists and policy makers have multiple reasons for grouping these air contaminants into specific categories. Categories may be created for pollution regulation purposed or based on how the contaminants affect health or the environment. Some contaminants may belong to several different categories, creating overlap and some categories may include a few contaminants, while others may include hundreds.

          

               Hand is the part of the body that contact with foreign substances most frequently.Our hand will constantly come in contact with foreign substances and hence,microorganism.Sweat from hand, food leftover on hand and dead skin cells is a very good nutrient for various microorganism eg:staphylococcus epidermis and Staphylococcus aureus.Since Sir Louis Pasteur had discover that bacteria is the cause of disease,the habit of washing hand proved important in preventing disease such as diarhea.Washing hand is a mandatory practices in hospital which show that hand play an important role in spreading bacteria.Furthermore,MRSA which is a more potent variant of Staphylococcus aureus which resist to normal anti-biotic medicine,hence more attention should be paid on hand cleanliness.

    

               The ear is exposed to the external/outside environment. Ear wax is composed mostly of dead skin cells and keratin with a small mixture of cerumen, sweat and oil. Cerumen is secreted from ceruminous gland which composed mainly cholesterol, wax esters, squalene, ceramides and triglycerides. The cerumen has antimicrobial properties which can be attributed to its slight acidic pH 5 and the presence of lysozyme. In normal circumstances, the ear wax is continuosly pushed out of the ear canal by slow migration of the top layer of skin cells from tympanic membrane towards the outer ear. The ear wax traps any foreign particles and microorganisms. Eventhough ceruminous gland has did a great effort to trap any foreign particles, but healthy outer ear still traps a various type of microbes. For examples, some common bacterias like Streptococcus saprophyticum, Staphylococcus epidermis, Turicellaotitidis, Alloiococousotitis, Corynebacterium, Staphylococcus aureus, and Pseudomonas aeruginosa. The common fungal microbes known to reside in the ear is Candida albicans. However, microbes that are known to inhabit in the middle ear are Streptococci, Haemophilus pneumoniae, and Mycobacterium.

Normal breathing is typically quiet without coughing, bubbling sounds or other noises. Human’s nose and throat contain a lot of microbes. Alveoli contain a lot of air sacs which is used for diffusion of air into the lungs. The surface of alveoli is moist so to increase the rate of diffusion of air. Moisture surface of alveoli contain microbes. Microbes will enter the nasal passage through inhalation from outside environment and out of nasal passage through exhalation. Most of the microbes  are non-pathogenic. Examples of pathogenic microbes are Haemophilus influenzae , Staphylococcus aureus, Streptococcus pneumoniae. Examples of non-pathogenic microbes are Neisseria, Streptococcus, Haemophilus, Micrococcus.

         Frequent coughing usually indicates the presence of a disease. Many viruses and bacteria benefit evolutionarily by causing the host to cough, which helps to spread the disease to new hosts. Most of the time, coughing is caused by a respiratory tract infection but can be triggered by choking, smoking, air pollution, asthma,gastroesophageal reflux disease, post-nasal drip, chronic bronchitis, lung tumors, heart failure and medications such as ACE inhibitors. A cough can be the result of arespiratory tract infection such as the common cold,pneumonia, pertussis, or tuberculosis. Infections in the breathing tubes can be caused by both bacteria and viruses, although the most common cause in children is a virus.Both gram-positive and gram-negative bacteria are commonly found in the throat, including a species of fungi (Candida). The predominant microbes found in the throat are alpha-hemolytic streptococci (viridans streptococci). The throat is a muscular passageway which carries food and liquids toward the digestive tract as well as air to the lungs. Because it encounters many different kinds of objects, it’s susceptible to encounters with many organisms. Throat's mucus funtion in facilitate food passage, but is also a way for microbes to latch on to epithelial cells and colonize. The throat is an excellent environment for microbes. Warm and moist, these ideal conditions provide aerobes and anaerobes an environment to flourish. However, the throat and tongue epithelial cells are constantly shedding, creating a complex environment that bacteria must adapt to in order to form or maintain a community. Violent couching is one of the way to release the microbes out of throat and mouth.

Precaution need to be taken when doing experiment:

1. Light up the flame to ensure the laminar flow on the surrounding.
2. Remove the cap and pass the neck of the bottle through the flame of a Bunsen burner to sterilize it when pouring agar.
3. Pass the neck of the agar bottle through the flame and recap the agar to avoid contamination of the agar.
4. Do not allow any agar to splash over the side of the plate.
5. We need to do movement with the shape of “8” for 5 times for the preparation of hand only.
6. We cannot pour hot agar into the plate with the sample of hand to avoid killing the bacteria inside of it.
7. An inoculated plate is always incubated in an inverted position to prevent condensation from falling onto the surface of the plate and interfering with discrete colony formation.
8. Sterilize all work areas and surfaces before and after handling the bacteria.

   Conclusion:            

           Microorganism such as virus, fungi, bacteria, algea and protozoa are present everywhere and dissimenate by different bioearosol especially wind or air stream. To prevent the spread harmful bacterias, proper cleaning of both hands and surfaces is very important. However, not all bacterisa are harmful; most bacterias are beneficial to us. Besides, when doing experiments, we should always use safe techniques to deal with bacterias. In this experiment, we have learnt that the different strains and morphologies of different bacterias.

    Reference:

         http://library.thinkquest.org/CR0212089/micr.htm

         http://suite101.com/article/normal-flora-and-opportunistic-pathogens-a93484

         http://spot.pcc.edu/~jvolpe/b/bi234/lab/differentialTests/ColonyMorphology.htm