Tuesday, 28 May 2013

LAB 6 : EXTRACTION AND MEASUREMENT OF PLASMID DNA USING GF-1 PLASMID DNA EXTRACTION KIT


INTRODUCTION
Every living cell contains DNA material that coded for its characteristic such us colour and cell function. There are several methods to isolate the DNA. DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding or sonicating the sample, removing  membrane lipids by adding a detergent or surfactants, removing proteins by adding a protease (optional but almost always done), removing RNA by adding an RNase (often done) and precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.
Plasmids, which are small molecules of DNA that can replicate independently of the chromosomal DNA. They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms.
          Plasmid is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs.
Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids and nucleic acids.  It is important to keep in mind the purpose of this procedure while performing it. The reason for this procedure is to separate the plasmid DNA from its associated proteins so that further manipulations can be done to it. Enzymes added to purify DNA in vitro can have unhindered access to it. These enzymes might be used for restriction mapping, ligation, sequencing, or other procedures to modify the DNA. A poorly purified plasmid preparation will only be partially accessible to the enzymes and this will cause many headaches in these later steps. For this reason, special care must be taken to ensure a pure DNA preparation.
A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95 °C) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations. Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has a ratio of 1.8 at 260/280 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

OBJECTIVE
To understand and practice proper technique of isolation process of plasmid DNA from bacteria.

MATERIALS AND REAGENTS
Centrifuge
Pipette
Incubator ( Digital Dry Bath )
Column
Microcentrifuge tube
Ethanol
Elution Buffer
Wash Buffer
Proteinase K
Buffer R1 and R2




PROCEDURES

Reminder :
* All steps are to be carried out at room temperature unless stated otherwise.
* Wash Buffer (concentrated) has to be diluted with absolute ethanol before use. Please
   refer to Reconstitution of Solutions .
* If precipitation forms in Buffer BG , incubate at 55°c - 65°c with occasional mixing until
   completely dissolved.

1. Centrifugation


1 mL of bacteria culture grown overnight or culture grown to log phase formed pellet    by centrifugation at 6000  x g for 2 min at room temperature. Supernatant was decanted completely.
Through removal of supernatant is essential as residual culture media may affect both yield and purity.




2. Re-suspension of pellet
100µL Buffer R1 was added to the pellet and the cells were re-suspended completely by pipetting up and down.
Ensure complete cell re-suspension. Lysis will not occur if clumps of bacteria remain following an inefficient re-suspension procedure.



3.Lysozyme treatment
10 µL lysozyme (50mg/mL) was added into the cell suspension of Gram-negative bacteria strains. While, for Gram-positive bacteria strains , 20 µL lysozyme (50mg/mL) was added into the cell suspension. They were then mixed thoroughly by vortex and were incubated at 37°C for 20 min.
Some bacteria strains may require longer incubation time in lysozyme.

4.Centrifugation
Pellet was formed when digested cells go through centrifugation at 10000 x g for 3 min. Supernatant was being decanted completely.



5.Protein denaturation
Pellet in 180 µL of Buffer R2 was re-suspended and 20 µL of Proteinase K was added. Vortex was used to mix thoroughly. It was then incubated at 65°C for 20 min with occasional mixing every 5 min.
Lysate should be clear at the end of incubation or else extend the incubation time to 30 min.

6.Homogenization
400 µL of Buffer BG which without RNase A treatment was added and mixed thoroughly by inverting tube several times until a homogeneous solution is obtained. It was incubated for another 10 min at 65°C.

 7.Addition of Ethanol
200 µL of absolute ethanol was added. The sample was mixed up immediately and thoroughly.
Mix immediately to prevent uneven precipitation of nucleic acid due to high local ethanol concentrations.

8.Loading to column
The sample was transferred into a column assembled in a clean collection tube.  Centrifugation at 10000 x g for 1 min was needed. After that, flow through was discarded.
In order to obtain maximum yield, we fix the orientation of the column during centrifugation at all time. We place the column which has a triangle mark on the edge, at a fixed position during centrifugation.
If column clogs, add 200 µL Buffer BG into column and centrifuged it.

9.Column washing
The column with 750 µL of Wash Buffer was washed and centrifuged at 10000 x g for 1 min. Flow through was also discarded.
Ensure that ethanol has been added into the Wash Buffer before use.

10.Column drying
The column undergo centrifugation at 10000 x g for 1 min to remove residual ethanol.
This step has to be carried out to remove all traces of ethanol as residual ethanol can affect the quality of DNA and may subsequently inhibit enzymatic reactions.

11.DNA elution
The column was placed into a clean micro-centrifuge tube. 100 µL of preheated Elution Buffer was added directly onto column membrane and stand for 2 minutes. DNA was eluted by centrifuged it at 10000x g for 1 minute and stored at 4°C or -20°C.
Ensure that the Elution Buffer is dispensed directly onto the center of the membrane for complete elution.   







RESULTS


DISCUSSIONS

There are some precaution steps to take when we are doing the experiment :
1) When re-suspend the cell completely by pipetting up and down, place the pipette on the same position to make 
    sure complete cell re-suspension.
2) When centrifuge , place the column which has a triangle mark on the edge to ensure that the process achieve
    optimization.


Problem Probability Suggestions
Low DNA yield Incomplete cell re-suspension Ensure that cells are completely re-suspended in Buffer R1, and Buffer R2 before incubation in lysozyme and Proteinase K.
Low elution efficiency Preheat Elution Buffer to 65 oC - 75 oC before eluting DNA
Low purity Incomplete protien denaturation Ensure that cells are completely re-suspended in Buffer R1, and Buffer R2 before incubation in lysozyme and Proteinase K. Extend incubation time until lysate clears.
RNA contamination Add RNase A to the sample as indicated in the protocol. Ensure that Rnase A used has not been repeatly frozen thawed. If necessary prepared a fresh stock.
Difficult to re-suspend cell pellet in Buffer R1 or R2 Centrifugation at high speed and long periods Ensure that the cell culture is centrifuged at the recommended speed and time.
Using pipette tips ,pipette the lysate up and down until completely homogenized.
Poor performance of eluted DNA in downstream application Eluted DNA contents traces of ethanol Ensure that the Column drying step is carried out prior to elution.

CONCLUSION

DNA is extracted from human cells for a variety of reasons. With a pure sample of DNA you can test a newborn for a genetic disease, analyze forensic evidence, or study a gene involved in cancer. Therefore, the practice on DNA extraction and measurement should be carried out carefully and precaution should be taken in order to get a pure DNA.

REFERENCES



Thursday, 23 May 2013

LAB 5 : Determination of Antimicrobial Effects of Microbial Extracts

Introduction:

     Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these antimicrobial compounds. Interest in naturally produced antimicrobial agents, such as bacteriocins, is on the rise, since nowadays consumers demand "natural" and "minimally processed" food.

     Bacteriocins comprise a large and diverse group of ribosomally synthesised antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive ad Gram-negative bacteria, those produced by lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural biopreservatives. Different classes of LAB bacteriocins have been identified on the basis of biochemical and genetic characterization. These bacteriocins have been reported to inhibit the growth of Escherichia Coli, Staphylococcus Aureus, and Salmonella.

Objective:

To determine the anitmicrobial effects of extracellular extracts of selected LAB strains.

Materials and reagents:

MRS broth
Sterile filter paper disk (50 mm X 50 mm)
Forceps
Sterile universal bottles
Cultures of LAB and spoilage/pathognic organisms
Bench-top refrigerated centrifuge
Incubator 30oC and 37oC
UV/Vis spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract

Procedure:

Part I. Determination of baceriocin activity via agar diffusion test:
          1. Label all the petri dishes according to the spoilage organisms and strains of LAB used.
          2. Each plate will be used for one strain of spoilage organism and one strain of LAB. Divide the plate
              into 2, each side for one replicate.
          3. Each group will have a strains of LAB and 3 strains of spoilage/pathogenic organisms.
          4. Load 10 mL of trypticase soy-yeast extract agar (TSAYE) into the labeled petri dishes and ensure
              that the agar covers the entire surface of the plate. Wait for it to solidify.
          5. Innoculate 2 mL of the broth containing the spoilage organism into 10 mL of brain heart infusion
              (BHI) agar and vortex.
          6. Load the mixture on top of the TSAYE agar layer, ensure that it covers the entire surface, and wait
              for it to solidify.
          7. Centrifuge the broth containing LAB cultures. The supernatant will be used as extracellular extracts.
          8. Aseptically pick up a sterile filter paper disk with your sterile forceps and dip the disk into the
              extracellular extract.
          9. Place the paper disk on top of the solidified BHI agar.
          10. Incubate the plates for 24 - 48 h at 37oC
          11. Upon incubation, measure the inhibition zones (in cm) and record your readings.

Part II. Determination of bacteriocin activity via optical density:
          1. Centrifuge the broth containing LAB cultures. The supernatant will be used as extracellular extracts.
          2. Each group will have a strain of LAB and 3 strains of spoilage/pathogenic organisms.
          3. Add 5 mL of double-strength MRS with 1 mL of cultures containing spoilage/pathogenic bacteria
              and vortex the mixture.
          4. Prepare a sterile dilution of the extracellular extracts. (Diluted 0x, 2x, 10x,50x, 100x)
          5. Add 5 mL of each extracellular extracts dilution into mixture as prepared in step (3).
          6. Incubate the mixtures for 12 - 15 h at 37oC.
          7. Prepare a control using 5 mL of double-strength MRS, 1 mL of cultures containing
              spoilage/pathogenic bacteria, and 5 mL of sterile peptone. Incubate the mixtures for 12 - 15 h at
              37oC.
          8. Prepare a negative-control for 'auto-zero' via the spectrophotometer. Add 5 mL of double-strength
              MRS with 2 mL of distilled water. (Need not incubate)
          9. Upon incubation, measure the optical density of the spoilage/pathogenic bacteria at 600 nm.
              Perform the same for the control as well.
          10. One arbitrary unit (AU) is defined as the dilution factor of the extracellular extract that inhibited
                50% of the spoilage/pathogenic bacteria growth and expressed as AU/mL.
          11. 50% of the spoilage/pathogenic bacteria growth are determined from the OD600 of the control.






Calculations:

Inhibition zone:

Strains of spoilage / pathogenic bacteria
Diameter 1 (cm)
Diameter 2 (cm)
Average diameter (cm)
E. coli
1.0
0.9
1.0
Salmonella
0.9
0.8
0.9
S. aureus
1.4
0.9
1.2

Arbitrary unit (AU/mL):


x: Serial dilutions of extracellular extract
y: Abs600 or OD600
m and c: constants

One arbitrary unit (AU) is defined as the dilution factor of the extracellular etract that inhibited 50% of the spoilage/ pathogenic bacteria growth and expressed as AU/mL.

Control: Abs600 = Z. Thus, 50% of Z = Z/2
y = mx + c ; Thus, x = (y - c)/m
When y = Z/2, thus x = (Z/2 - c)/m





Graph :








Discussion:

Part 1 :

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

   1.  Before we pour the 2mL of the broth containing the spoilage organism inoculate into 10mL of 
         brain hear infusion ( BHI ) agar,vortex centrifugation is needed. Avoid direct poured of the 
        mixture into the Petri dish that contain TSAYE agar and mixed it with  "8" number motion .

   2.  Do not cover the petri dish immediately to prevent the trapping of water vapours which
        released from hot tryticase soy-yeast extract agar ( TSAYE ) agar to prevent the formation of
        water droplets in order to maintain the growth rate of bacteriocins.

   3. The Escherichia Coli and Salmonella were not inhibited by the LAB strain successfully. The
        specimen of pathogenic bacteria provided contain live Escherichia Coli and
        Salmonella.
  
     Part 2 :
      
          Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer Is a photometer ( a device for measuring light intensity ) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement. Spectrophotometers are commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 420 nm- 660 nm using different controls and calibrations. Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination.
      
      1. The results showed that the control sample value, OD600 is less than the extracted
          values because lactic acid and other types of acid were existed in the extracted sample.
          Thus, it caused the growth of the bacteria in it while peptone which found in the control
          sample only has nutrient and without any other promoting factors.


Conclusion:

 In conclusion, the diffusion of agar method showed that the strain of LAB against the S.aureus , a
 pathogenic bacteria most effectively. Moreover, the S.aureus in the second part of the experiment also
 showed the best inhibition but the LAB does not showed the strongest inhibition effects on the S.aureus
 pathogenic bacteria due to the technical error in the preparation of the sample of OD600.


Reference:



Wednesday, 1 May 2013

LAB 4 : Sources of Contamination and Infection

Introduction :

     Airborne micro-organisms are usually carried on dust particles, although some ( fungal spores, for example ) ay be carried directly by air currents. It is important for microbiologist to be aware of the potential for contamination by airborne micro-organisms. Carefully observation of simple precautions dramatically reduces the risk of contamination of the cultures.

     Every human is colonized by billions of micro-organisms. These micro-organisms, some of which are vital to our well-being, constitute our resident or normal microflora. Resident micro-organisms are nourished by the chemicals and moisture excreted by the human bod. In moist areas such as armpit, there may be one billion bacteria per cm^3 ; on the drier skin of the forearm, there may be 10000 bacteria per cm^3!!!

     Resident micro-organisms are either non-pathogenic or are prevented from infecting the body by an array of mechanical and chemicals defences. Some resident microbes are, however, opportunistic pathogens which may cause infection if the body's defences are breached-for example, if the skin is broken.

      Transient micro-organisms are picked up from our environment- for example from faecal contact or from soil-and usually fail to become permanent skin residents. One of the most important reasons for failure to gain permanence is that the established residents are better able to compete for nutrients, Since transients generally originate in other environments, they are poorly adapted to conditions on the skin and usually disappear within 24 hours arrival.

     Micro-organisms in the upper respiratory tract are either normal residents or transients, As with the skin, normal microflora largely consists of non-pathogens or opportunistic pathogens.

     Large numbers of transients enter the upper respiratory tract as we breathe or eat. They may also come from our own hands or from improper sanitation during food preparation. Regardless of their origin, most transients are non-pathogenic and are quickly killed by various defences arrayed against them.

Objective :

To determine the micro-organisms in the air and from healthy humans.

Materials and reagents :

Molten nutrient agar
Sterile water
sterile Petri dishes
Sterile clinical swab
Pipette and tips

Procedure :

Air :
         1. Pour the molten agar into sterile Petri dish and cool.
         2. Remove the lid from the plate and leave it resting on the side of the plate, facing
             down. ( Never invert the lid of the Petri dish). Leaves the plates exposed for 5
             minutes.
         3. Replace the lids and incubate at 37oC for 48 hours.

Hands :
         1. Wash hand using sterile water. Do not use soap.
         2. Use an automatic pipette to transfer 1mL of wash water to the Petri dish.
         3. Add molten nutrient agar to the Petri dish.
         4. Replace the lids of the Petri dish and gently rotate the dish until the wash water is
             thoroughly mixed with the molten agar. Do not allow the agar to contact the lid of the
             dish.
         5. After the agar has set, invert the dish and incubate at 37oC  for 48 hours.

Ear :
         1. Pour the molten agar into the sterile Petri dish and cool.
         2. Using extreme care, rub the sterile swab moistened with sterile isotonic solution into
             the ear of the subject.
         3. Use the swab to inoculate the labelled plate. Distribute the inoculum as in the streak
              method.
         4. Incubate at 37oC  for 48 hours.

Normal breathing :
         1. Pour the molten agar into the sterile Petri dish and cool.
         2. Remove the lid and hold the plate about 15cm from your mouth. Breathe normally
             but directly onto the plate for one minute. Replace the lid.
         3. Incubate at 37oC  for 48 hours.

Violent coughing :
         1. Pour the molten agar into sterile Petri dish and cool.
         2. Remove the lid and hold the plate 15cm from your mouth. Cough violently onto the
             agar. Replace the lid.
         3. Incubate 37oC for 48 hours.

Results :


a) Using nutrient (commercial) agar : 



1) Air colonies


Form
Irregular, Filamentous
Elavation
Raised, Umbonate
Margin
Curled

2) Cough colonies

Form
Circular, Irregular, Filamentous
Elavation
Raised
Margin
Curled


3) Breath colonies


Form
Circular, Irregular
Elavation
Raised, Umbonate
Margin
Entire

4) Ear Colonies


Form
Circular, Irregular
Elavation
Raised, Flat, Crateriform
Margin
Entire, Filiform

5) Hand Colonies


Form
Circular, Irregular
Elavation
Flat
Margin
Entire



b) Using Own Recipe :

1) Air Colonies 


Form
Circular, rhizoid
Elavation
Raised
Margin
Curled


2) Hand Colonies


Form
Rhizoid, irregular, circular
Elavation
Umbonate, crateriform, raised, flat
Margin
Entire, lobate
3) Ear Colonies


Form
Circular, irregular
Elavation
Convex, raised
Margin
Filiform, entire
4) Breath Colonies


Form
Circular, irregular
Elavation
Convex, flat, umbonate
Magin
entire
5) Cough Colonies


Form
Filamentous
Elavation
Crateriform,  umbonate, flat, raised
Margin
Entire,  undulate







Discussion :
1. When pour the nutrient in to the Petri dish, remember mix well the solution. This is to
    avoid the agar from deposit at the bottom of the solution which may result the agar being
    solidify at the bottom and causes the nutrient in the Petri dish cannot be solidify.

2. The nutrient must keep at certain temperature to avoid the agar from being solidify.
    When see the bottom of the agar start to solidify, put the agar solution in the oven and
    reheat it.

3. When pour the nutrient avoid the nutrient touching the lid or side of the Petri dish.
    Because this may cause contamination that will affect your result.

4. When doing the each contaminant sample, remember to cool down the temperature of the
    nutrient solution until the temperature that are touchable by our hands. This is to avoid
    the bacteria killed due to the high temperature of the nutrient and affected the result.

5. After pour the nutrient into the hands` sample remember to mix the hand washed water
    and nutrient agar by doing 5 times “ 8 “ number motion slowly to mix it well.



Conclusion :
Micro-organism like fungi, bacteria and protozoa can be found in air and surrounding environment that compatible to their living condition. micro-organism also can live within living host like us for example in our hand and some of them might cause harm if a proper technique of hang washing is ignore. but most of them are symbiont living thing without causing any harm or good.

Reference :

http://en.wikipedia.org/wiki/Infection

http://en.wikipedia.org/wiki/Contamination

http://en.wikipedia.org/wiki/Microorganism#Habitats_and_ecology

http://wiki.answers.com/Q/Do_we_have_bacteria_on_our_hands#ixzz1IFqPqcDp

http://www.articlesbase.com/diseases-and-conditions-articles/types-and-prevention-of-bacteria-471084.html