LAB REPORT 6 (BACTERIAL DNA EXTRACTION)

INTRODUCTION

           DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. DNA is extracted from human cells for a variety of reasons. With a pure sample of DNA we can test a newborn for a genetic disease, analyze forensic evidence, or study a gene involved in cancer.

            Gram-negative bacteria are a group of bacteria that do not retain the crystal violet stain (when rinsed with alcohol) used in the Gram staining method of bacterial differentiation,^[1] making positive identification possible. It has thin layers of peptidoglycan and the bright pink to red colour will appear when it stained with safranin. The example of gram-negative bacteria are Pseudomonas sp. and E.coli sp.

              Gram-positive bacteria are bacteria that give a positive result in the Gram stain test. Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-coloured when seen through a microscope. It also has thick layer of peptidoglycan in cell wall and less lipids. The peptidoglycan traps the crystal violet in the cytoplasm. The violet stain also is not washable even by using alcohol. The example of gram-positive bacteria are Staphylococcus sp. and Bacillus sp.

                In the experiment, centrifugation method played the main role and did used for few times. A centrifuge is a laboratory device that is used for the separation of fluids, gas or liquid, based on density. Separation is achieved by spinning a vessel containing material at high speed; the centrifugal force pushes heavier materials to the outside of the vessel.

Objective

·         To extract DNA from different organisms.

·         To determine the purity of DNA.

·         To determine the best ratio of purity.

·         To differentiate DNA of gram-positive bacteria and gram-negative bacteria.

Procedure:

1. Centrifugation

1 ml of bacteria culture grown to log phase was turned to pellet by centrifugation at 6000 x g for 2 min at room temperature. The supernatant was decanted completely.

2. Resuspension of pellet

100µl Buffer R1 was added to the pellet and the cells were resuspended completely by pipetting up and down.

3. Lysozyme treatment

For Gram-negative bacteria strains, 10 µl lysozyme (50mg/ml) was added into the cell suspension. For Gram-positive bacteria strains, 20 µl lysozyme (50mg/ml) was added into the cell suspension. They were mixed thoroughly and incubated at 37°C for 20 min.

4. Centrifugation

Digested cells were centrifuged at 10000 x g for 3 min and pellet was formed. The supernatant was decanted completely.

5. Protein denaturation

Pellet was resuspended in 180 µl of Buffer R2 and 20 µl of Proteinase K was added. It was mixed thoroughly. Then, it was incubated at 65 °c for 20 min with occasional mixing every 5 min.

6. Homogenization

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

7. Addition of Ethanol

200 µl of absolute ethanol was added. It was mixed immediately and thoroughly.

8. Loading to column

The sample was transferred into a column assembled in a clean collection tube.it was centrifuged at 10000 x g for 1 min. Flow through was discarded.

9. Column washing

The column was washed with 750 µl of Wash Buffer and was centrifuged at 10000 x g for 1 min. Flow through was discarded.

10. Column drying

The column was centrifuged at 10000 x g for 1 min to remove residual ethanol.

11. DNA elution

The column was placed into a clean microcentrifuge tube. 100 µl of preheated Elution Buffer,TE buffer was added directly onto column membrane and was allowed to stand for 2 min. It was centrifuged at 10000 x g for 1 min to elute DNA.

Reminder:

1. All steps are to be carried out at room temperature.

2. Wash Buffer (concentrate) has to be diluted with absolute ethanol before use.

3. If precipitate forms in Buffer BG, incubate at 55°c -65°c with occasional mixing until completely dissolved

Results

Bacteria	260	280	Ratio=260/280
Lactobacillus	0.273	0.190	1.437
Lactobacillus	0.151	0.127	1.189
Salmonella	0.318	0.210	1.514

Discussion

Gram-negative cell is bacterial cell having wall composed of a thin peptidoglycan layer of their cell wall It consists of a triple layer and outermost layer contains lipopolysaccharide (LPS). Salmonella is gram-negative cell.

Gram-positive cell is bacterial cell having a thick wall of peptidoglycan layer and it can retain the crystal violet stain in the gram staining method. Lactobacillus used in this experiment are Gram-positive cell.

Lysozyme is enzyme that break down bacterial cell walls to improve protein or nucleic acid extraction efficiency. The enzyme acts by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins.

Ethanol precipitation is used to purify DNA and polysaccharides from aqueous solution by adding ethanol as an anti-solvent. Since the DNA insoluble in ethanol but soluble in water, so the ethanol can removes some of the salts present in the leftover supernatant.

The purity of DNA can be calculated by formula below:

Purity of DNA = OD_{260 /} OD₂₈₀

Ratio between the readings at 260 nm and 280 nm (OD260/OD280) provides an estimate of the purity of the sample. The best ratio of the purity is between 1.7-1.9.

Based on the result observed, the ratio of the purity is not lie in the range between 1.7 and 1.9. This is due to several factors as below:

Factors	Possibilities
Incomplete cell suspension	Inefficient of cell suspension during the experiment carried out.
Low elution efficiency	Elution buffer does not dispensed directly onto the center of the membrane.
Contamination	Supernatant removed incompletely and still stick with the fluid.

Conclusion

DNA extraction of Gram-negative bacteria is much harder than DNA extraction of Gram-positive bacteria. Precaution steps should be conducted to get the pure DNA.

Reference

http://learn.genetics.utah.edu/content/labs/extraction/howto/

http://www.life.umd.edu/classroom/bsci424/BSCI223WebSiteFiles/GramPosvsGramNeg.htm

http://www.highveld.com/microbiolog

LAB 5: Determination of Antimicrobial Effects of Microbial Exracts

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, antibacterial are used against bacteria and antifungals are used against fungi. A wide array of antimicrobial classes that are used for animal health and production are also used for human treatments. Those antimicrobials genes that propagate from the use of antibiotics for animal populations may spread and eventually hurt the efficacy of these same antibiotic when they are used to treat people.

               While all antimicrobial is a potential human health hazard, the preserved efficacy of some human medicine antibiotics is of higher priority. Antimicrobial resistance is only a threat to public health when humans are infected with a resistant organism that is difficult or impossible to treat. This is an issue seen more frequently with human pathogens transmitted between humans (such as extremely drug resistant tuberculosis, also called XDRTB, and MRSA). While outbreaks of resistant foodborne pathogens have been reported, very few have been epidemiologically traced back to the farm. Even fewer have been specifically associated with a specific indication such as increased rate of gain or prevention of a particular disease.

             Bacteriocins comprise a large and diverse group of ribosomally synthesized antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive and 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 Listeria monocyotogenes, Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.

Materials and reagents

·         MRS broth

·         Sterile filter paper disk

·         Forceps

·         Sterile universal bottles

·         Cultures of LAB and spoilage/pathogenic organisms

·         Bench-top refrigerated centrifuge

·         Incubator 30oc and 37oc

·         UV/V is spectrophotometer

·         Distilled deionized water

·         Trypticase soy agar

·         Brain heart infusion agar

·         Yeast extract

Procedure

Part I: Determination of bacteriocin activity via agar diffusion test

1.      Three petri dishes were labelled for each group.

2.      A petri dish was shared between two person.

3.      Each petri dish was divided into four parts and each side for one replicate.

4.      A strain of pathogen (Staphylococcus aureus) and a strain of lactic acid bacteria (LAB) were used in this experiment.

5.      10 ml of trypticase soy-yeast extract agar (TSAYE) was added into each labelled petri dish and the petri dish was moved in “8 shape” on the work surface to ensure the agar was distributed evenly.

6.      The petri dish was left aside for solidification.

7.      2 ml of pathogen (Staphylococcus aureus) was innoculated into 10 ml of brain heart infusion (BHI) agar.

8.      The innoculation process must be carried out quickly to avoid solidification of BHI agar.

9.      The mixture is then loaded on top of the TSAYE agar layer and the petri dish was moved in “8 shape” on the work surface to ensure the agar was distributed evenly.

10.  The petri dish was left aside for solidification.

11.  50 ml of broth containing LAB cultures was centrifuged at 3500rpm for 10 minutes. The supernatants will be used as extracellular extracts.

12.  The forceps were held by its handle and the tip of the forceps were heated with the flame of a Bunsen burner.

13.  A sterile filter paper disk was aseptically picked up using sterile forceps.

14.  The paper disk was then dipped into Lactobacillus Fermentum containing extracellular extracts. The excess extract was drained off completely.

15.  The paper disk was placed on top of the solidified BHI agar.

16.  The petri dishes were incubated for one day at 37°C.

17.  Upon incubation, the inhibition zones were measured (in cm) and the readings were recorded.

Part II: Determination of bacteriocin activity via optical density

1.      Universal bottles were prepared.

2.      Each of the universal bottle was labelled with 0x, 2x, 10x, 50x ,100x and control.

3.    Lactobacillus Plantarum was mixed with MRS to be diluted to final volume of 5 ml in each universal bottle labelled. The volume of the Lactobacillus Plantarum and MRS needed for each universal bottle is shown as below:

	0x	2x	10x	50x	100x	control
Volume of Lactobacillus Plantarum (ml)	5.0	2.5	0.5	0.1	0.05	0
Volume of MRS (ml)	0	2.5	4.5	4.9	4.95	5.0
Total (ml)	5.0	5.0	5.0	5.0	5.0	5.0

4.     Each of the mixture was then added with 5 ml of MRS and 1 ml of pathogen Escherichia coli (E.coli).

5.      The universal bottles were then incubated for 12-15 hours at 37°C.

6.    Upon incubation, the optical density of the pathogenic bacteria in each universal bottle was measured at 600 nm using spectrophotometer.

Results:

Part 1: Determination of bacteriocin activity via agar diffusion test.

Strains of LAB: Lactobacillus plantarum

Strain of LAB	Strain of pathogenic bacteria	Inhibition zone (cm)
Lactobacillus Fermentum	Staphylococcus aureus	Diamater1 (cm)	Diameter 2 (cm)	Average diameter (cm)
		0.95	0.80	0.88
		0.60	0.70	0.65
		0.73	0.76	0.75
		0.65	0.64	0.65
		0.52	0.45	0.49
		0.50	0.30	0.40

Part II: Determination of bacteria activity via optical density

Dilutions	OD600 of spoilage/ pathogenic bacteria
	Strain 1 : E.coli
0x	0.254
2x	0.384
10x	0.351
50x	0.349
100x	0.428
Equation	y=0.00098x+0.33
OD600 of control	0.1370
50% of OD600	0.0685
AU/ ml	-266.84

DISCUSSION

Part 1: Determination Of Bacteriocin Activity Via Agar Diffusion Test

1.      Bacteriocin are proteinaceous toxins produced by bacteria to inhibit the growth of microorganisms such as fungi, protozoans and similar or closely related bacterial strains by producing intraspecies antagonistic effects.

2.      The larger the inhibition zone (no bacteria growing area) on the agar medium, means that the bacteriocin is effective on the pathogenic bacteria and vice versa.

3.      The production of bacteriocins, organic acids, free fatty acids, ammonia, diacetyl and hydrogen peroxide by lactic acid bacteria (LAB) promotes bacterial interference. These metabolites have been applied for many years to extend the shelf life of foods in food industry by enabling acidification that will inhibit the growth of spoilage agents. Besides that, proteinaceous bacteriocins produced by LAB supress the growth of pathogenic microorganisms.

4.      As for none inhibition zone exist result are because not enough Lactobacillus fermentum are being applied around the pathogenic bacteria. This is because without adequate numbers of Lactobacillus fermentum, the point of critical mass which is needed cannot occur and the bacteria will be unable to have the desired impact on the symptoms being treated.

Part 2: Determination Of Bacteriocin Activity Via Optical Density

1.      The optical density (OD) or called as scattering intensity is the measure of transmittance of an optical medium for a given wavelength. The higher the OD the lower the transmittance, the higher protection factor by a filter (e.g. goggles, viewing windows, etc.). This factor allows us to calculate the cell density from any measured OD. A spectrophotometer can be set at a wavelength of 420 – 660 nm. In this experiment, the OD600 is measured. Typically, this wavelength must be standardized and may need to be adjusted specifically to the material being tested. Different vegetative cells and bacterial spores may not have the same maximal absorbance wavelength.

2.      One arbitrary (AU) is known as the dilution factor of the extracellular extract that inhibited 50% of the spoilage or pathogenic bacteria growth and expressed as AU/mL.

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

3.      The positive control which showed the growth of bacteria without extracellular extract of lactic acid bacteria has been set up for each pathogenic bacteria. TheOD600 of the positive control was then measured in order for us to investigate whether there is inhibition of pathogenic bacteria activity by comparing the OD600 of the samples. If the OD600 of the sample is less than OD600 of the positive control, there will be inhibition of spoilage bacteria.

4.      Based on our result, LAB shows low inhibition activity on E.Coli. This might be due to we didn’t shake the bottle of bacteria culture before pipetting especially for the positive control. Therefore, the reading for positive control is lower than all the samples of different dilution.

CONCLUSION

Some of the microorganisms can synthesis certain substances that have antimicrobial effects. Bacteriocins produced by lactic acid bacteria (LAB) as bio preservatives against both Escherichia coli (E.coli) and Staphylococcus aureus. Lactic acid bacteria (LAB) also synthesize bacteriocins that have antimicrobial effects. The use of strains that produce multiple bacteriocins could be advantageous to limit the potential emergence of bacteriocin-resistant populations.

REFERENCE

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

http://www.medicinenet.com/staph_infection/article.htm

http://www.microbiologybytes.com/LabWork/bact/bact14.htm

http://www.mpi-bremen.de/Binaries/Binary5381/Wachstumsversuch%283%29.pdf

http://www.biology-online.org/dictionary/Optical_density