To isolate bacteria colonies using spread plate and serial dilution technique

Objectives: To isolate bacteria colonies using spread plate and serial dilution technique.

Introduction:
A spread plate was different from a drop or pour plate, the basic process required spreading the bacteria over the agar plate in order to separate the bacteria colonies and allowed to easily view the differences in between colonies such as size, shape, elevation, texture, light transmission and colony pigmentation. The spread plate was also useful for determining the concentration of the original mixture. Individual bacterial were very difficult to see when spread concentrated solution of bacterial culture on agar surface, the serial dilution were used to dilute the amount of bacterial culture in each agar plate, so the last plate should have small amount of bacteria, isolated colonies that can be easily counted or studied. A good method of spread plating involves the use of the glass rod bent in an “L” shape. The micropipette can be used to transfer the amount of cell culture onto the surface of agar in the plates. Once the amount of cell culture is transferred on the center of the agar on the plates, the L-rod can be used to spread the liquid across the surface. Colonies should then grow comfortably across the entire plate surface. A colony of bacteria is made up of millions of identical bacterial cells each original from one single cell.
Materials:
Micropipettes P200, P1000 with rack, microfuge tubes with rack, micropipette disposable tips, tips disposable cup, acetone burner, lighter, agar plates, L-rod, alcohol breaker, ethanol (70% EtOH), sterile distilled water (dH2O), water bath, Vortex genie and stock bacterial culture tube.
Methods:
Labeled the bottom of each agar plate with name, date, culture name and 1 (1:10), 2 (1:100), 3 (1:1,000), 4 (1:10,000), 5 (1:100,000), 6 (1:1,000,000). Labeled microfuge tubes with the number and diluted ratio above. Set micropipette P1000 to the volume desired 900 μl, inserted disposable tip, lighted acetone burner, opened sterile dH2O cap, passed the lip and cap through the flame, and then dispensed 900 μl to all six microfuge tubes and then discarded tip to the disposable cup. Set micropipette P200 to volume desired 100μl, then took stock bacterial culture tube to vortex well, inserted disposable tip, opened stock bacterial culture tube, passed through the  flame and transferred 100μl from stock bacterial culture to tube 1 and took it to vortex well, then opened tube 1 and passed through the flame and used the same micropipette P200 to transfer 100μl to tube2. Applied the same steps as tube 1 to tube2, tube 2 to tube 3, tube3 to tube 4, tube 4 to tube 5 and tube 5 to tube 6 using the same volume 100μl to dispense. Filled little alcohol into the breaker, opened tube1 and used same micropipette P200 to transfer 100μl from tube 1 to agar plate 1 by left hand hold the agar plate and opened good enough for pipetting while right hand hold micropipette, then took the L-rod to dip into alcohol in breaker and passed through the flame, let cool down, and then spread lightly on agar surface, made sure to spread cover all the agar surface, took out the L-rod and closed the lid at the same time, dipped the L-rod in alcohol and passed through the flame, then sealed with parafilm and put it upside down on other table. Applied the same steps on agar plate 1 to finished plate 2, 3, 4, 5, 6 labeled on the plates, made sure tube 2 to plate 2, tube 3 to plate 3, tube 4 to plate 4, tube 5 to plate 5 and tube 6 to plate 6.  After finished all six sample plates, placed all upside down into water bath and set 37°C temperature, allowed to incubate about 16 to 18 hours. Compared the sample plates from plate 1 to plate 6 to identify and isolated colonies between each plate.
Results / Calculations:
Prepared 1ml of 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000 serial dilution using bacterial culture stock solution.
Figure: The different between each bacterial culture plates after incubation.
The number of bacteria was reduced by dilution before the bacterial culture was spread on the surface of the nutrient agar plate after 16 to 18 hours incubation, the culture plate #6 had more bacterial isolated colonies than other culture plates. The culture plates #1, 2, 3, and 4 did not have any bacterial isolated colony; culture plate #5 had some isolated colonies. The bacterial isolated colonies were small circular size, margin entire, elevation raised, texture smooth, translucent and white color.
Conclusion / Discussion:
Successfully learned how to use the technique of spread plate and serial dilution to isolate individual bacterial colonies on a nutrient agar plate. The basic of obtaining isolated colonies were reduction the number of bacteria per unit volume by dilution. The culture plate #6 had higher number of bacterial isolated colonies than other culture plates. It is important when prepared to make the serial dilution, make sure to mix well the culture stock solution before use to dispense.

References:
Basic Biotechnology Laboratory Manual by BioHealth College, Inc.

DNA Fingerprinting

Objectives:  To learn the restriction digestion and staining DNA sample.

Introduction: DNA fingerprinting is a very quick way to compare the DNA sequences of any two or more living organisms. DNA fingerprinting can be used to trace the inheritance of genetic disorders, identify origin of a blood or criminal investigation. DNA is a linear, polymeric molecule composed of four different nitrogenous bases: Adenine, cytosine, guanine and thymine. Restriction enzymes are used to cleave the DNA at specific sites based on nucleotide sequences, the common restriction enzymes are EcoR I (GAATTC), Pst I (CTGCAG) and Hind III (AAGCTT). The technique of DNA fingerprinting requires that the DNA be cut up into small fragment using the restriction digestion of DNA samples. DNA is naturally colorless, hard to visible in the gel without UV light. Fast Blast DNA stain can be used as an in-gel stain, by incorporating it into an agarose gel and electrophoresis buffer so that it stains DNA while it is running through the gel. Fast Blast DNA stain molecules strongly bind to the DNA fragments to become visible without the UV light.

Materials: Micropipette and tips, disposable tips cup, 1.5ml microfuge tubes and rack, Lambda DNA stock with labeled S1, S2, S3, S4 and S5, crime scene (CS), EcoR I/Pst I enzyme mix, foam tube holder, water bath incubator, loading dye, 1% agarose, weigh paper, digital analytical balance, microwave, 250ml flask, graduated cylinder, paper towel, TAE buffer, 100X and 500X Fast Blast stain, gel box, gel tray, gel comb, warm water and staining tray.

Methods: Labeled five 1.5ml microfuge tubes with initial and suspect S1, S2, S3, S4 and S5. Used a new micropipette tip for each sample, transferred 10μl of each stock DNA sample from stock S1 to tube S1, stock S2 to tube S2, stock S3 to tube S3, stock S4 to tube S4 and stock S5 to tube S5 labeled. Used a new micropipette tip for each sample, added 10μl of the Enzyme Mix (EcoR I/ Pst I) to each tube and mixed well with pipette. Made sure all the tube caps tighten, put in a foam tube holder and placed in water bath incubator at 37°C for 45 minutes. After incubation, took out and used new micropipette tip for each sample, pipetted 5µl of loading dye to each tube and mixed gently with pipette plunger. Stored in refrigerator at 4°C temperature (Long-term storage should be at -20°C). Run gel electrophoresis to detect the presence of DNA using Fast Blast stain. Weighed out 0.5g of agarose with weigh paper on digital analytical balance, poured into a 250ml flask, used graduated cylinder to measure 50ml of TAE buffer poured in a flask to mix with agarose and covered with paper towel, then put it in microwave, heated about 90 seconds until all agarose dissolved, allowed to cool down between 50°C - 55°C under the hood, added 33µl of 500X Fast Blast to the gel. Set up gel box and the comb in place. Poured the gel slowly into the gel tray, pushed any bubbles away to the side using pipette tip. Let the gel solidified. Removed the comb from the gel and placed gel tray into the gel box, then filled TAE buffer to the gel box until the gel covered with buffer, added 200µl of 500X Fast Blast per 300ml of 1X TAE buffer in gel box. Used a new tip for each sample, loaded 12µl for each sample (CS, S1, S2, S3, S4, S5) into the wells and run gel for 30 minutes. After electrophoresis was completed, turned off the power and removed the gel tray out and carefully slide it into the staining tray to stain the gel with 100X Fast Blast stain for 2 minutes, then transferred the gel into other tray with warm tap water (40° - 55°C). Gently shake the gel in water to wash, repeated to change warm water about 5 times, until the dye no more came out from the gel. Placed the gel on a light background and recorded the results.

Results/Calculations: To prepare to make 50ml of 1 % agarose:
                    1 g   x  50ml = 0.5 g of agarose
                   100ml
Picture: Showed the results between crime scene and suspects DNA fragments in 1% agarose gel with Fast Blast stain.
The well suspect (S3) DNA bands lined up with crime scene (CS) DNA bands, suspect S1, S2, S3, S5 had one band matched to CS. The well suspect S4 was no DNA matched with CS.
             
Conclusion/Discussion: The same restriction enzymes mix (EcoR I/ Pst I) added to Crime Scene and each Suspect DNA samples, DNA fragments were successfully visible in 1% agarose gel with the Fast Blast stain on a light background. The Suspect S3 appeared to be the guilty party, because the DNA bands matched to the Crime Scene. The well S4 was not successfully digestion, may cause by either DNA sample incomplete digestion, DNA sample incubated too long or DNA sample was contamination. Fast Blast DNA stain is nontoxic and noncarcinogenic, but it will stain skin and clothing. Wear gloves and a lab coat whenever handling stain solutions or stained gels.

References:
Advanced Biotechnology Laboratory Manual by BioHealth College, Inc.
http://learn.genetics.utah.edu/content/labs/extraction/

To detect the presence of antigen in the sample with ELISA method.

Introduction:  
ELISA stands for enzyme-linked immunosorbent assay, also known as an enzyme assay, is used to test for many diseases by detecting either the antibody or the antigen. ELISA is a powerful diagnostic tool in human medicine, disease detection, animal, plants, detecting illegal drug used, and testing indoor air quality and food safety. A sample contains many proteins and may or may not contain the antigen, the two unknown antibody will bind to the antigen if presence in a sample, the last was to evaluate the assay results after enzyme substrate added to the wells. Positive and negative controls are used to compare to the test well. The positive control has a strong color change; the negative control has little or no color change.
Materials: Micro plate, multi channel pipette, single channel pipette, micropipette tips, tips disposable cup, ELISA wash buffer, paper towels, reagent reservoir, positive control, negative control, primary antibody (1°Ab), secondary antibody (2°AB), enzyme substrate, unknown sample A and sample B.

Methods: Labeled micro plate first row 12 wells in order with ++--AAAABBBB. Used new tip with each type of sample, loaded 50μl from positive sample to + wells, negative sample to – wells, A sample to A wells and B sample to B wells labeled. Waited 5 minutes and flipped over the micro plate, then gently tapped on the paper towel, made sure to avoid splashing sample back into wells. Used multi channel pipette to load 150μl of ELISA wash buffer to each well, then flipped over the micro plate and gently tapped on the paper towel. Repeated 1 more time. Used new tip to pipette 50μl of 1°Ab to each well and waited 5 minutes, then flipped over on the paper towel let the liquid out. Used the same step as above to wash 2 times with 150µl of wash buffer. With a new tip to load 50µl of 2°Ab to each well and waited 5 minutes, then poured out and gently tapped on paper towel. Used the same step as above to wash 3 times with 150µl of wash buffer, and then used a new tip to loaded 50µl of enzyme substrate to each well. Viewed the results for the presence of the antigen.

To streak plate and bacterial culture transfer using aseptic technique.

Introduction:
Aseptic technique is a laboratory technique that is required to transfer pure cultures and performed under sterile conditions to prevent contamination of the culture by unwanted microbes. Bacterial culture streaking allowed bacteria to reproduce on a culture medium in a controlled environment. The process streaked bacteria in four section on an agar plate and sealed with parafilm and allowed to incubate at 37°C temperature for about 16 to 18 hours. Bacterial culture streaking can be used to identify and isolate pure bacteria into individual colonies. Microbiologists use bacterial and other microbial culture streaking methods to identify microorganisms and to diagnose infection.

Materials:
Agar plate, bacteria culture slant, acetone burner, lighter, inoculating loop, parafilm and Digital constant temperature tank (water bath).

Methods:

1. Labeled the bottom of agar plate with name, date, culture name and labeled number 1, 2, 3, 4 counter clockwise, each across ¼ of the plate.
2. Lighted acetone burner with lighter, flamed the inoculating loop tip until it turned orange color, then passed the loop wire to flame, and let it cool down.
3. Opened the bacteria culture slant cap by right hand hold the inoculating loop and the cap of the culture slant while left hand hold and loosen the culture slant, passed the lip through the flame, and then dipped the loop into the culture slant to transfer the bacteria to a new agar plate, tighten the cap of the culture slant, then opened the lid of the agar plate enough for the loop in while other hand put the inoculating loop that had bacteria culture sample to section 1 labeled on the plate to transfer bacteria sample by streaked the agar surface lightly back and forth several time, then closed the plate, turned the culture plate to section 2 and flamed the loop at the same time, and allowed to cool down, then opened the lid plate, used the inoculating loop to streak on section 2 several times overlap the end of section 1, closed the plate, and turned the plate to section 3, and flamed the loop at the same time, and allowed to cool down, and used the same steps to finish streaking in section 3 and 4.
4. The different of section 4 only streaked one line overlap the end of section 3, and made sure did not touched other section, then flamed the loop and allowed to cool down and put it on the table. Flipped the culture plate and sealed with parafilm, opened the top cover of the water bath, put the bacteria culture plate upside down in the water bath, made sure had enough water, then closed the cover and set 37°C temperature, allowed to incubate about 16 to 18 hours.
5. Viewed the result to identify and isolated colonies from the bacteria culture slant.

To extract and isolated DNA from Human cheek cells and run gel electrophoresis.

Introduction:
All living organisms contain DNA. DNA extraction is a basic procedure of isolating the DNA from other cellular substances. It is also useful for the detection of bacteria and viruses from the environment. A pure sample of DNA that extracted from human cells can be used to test for a genetic disease, study a gene involved in cancer, body identifications or analyze forensics. Chelex resin beads used to remove the ions out of the DNA sample to prevent ions interfered with the PCR reaction. The DNA in cheek cells is identical to the DNA in blood cells.

Materials:
Part A: 0.9 % Sodium Chloride (NaCl), a 1.5ml microfuge tube #2 containing 200μl of 5% Chelex resin beads, paper cup, 50ml centrifuge tube capped with rack, 1.5ml microfuge tubes with rack, micropipettes and tips, serologital pipettes and electric pipettor, balanced centrifuge, vortex mixer, water bath incubator, ice bath.
Run gel: 0.8% agarose, flask, paper towel, graduated cylinder, loading dye, Ethidium Bromide (EtBr), micropipette and tips, gel tray, gel box, parafilm, microwave, digital analytical balance, TAE buffer, paper towel, Cheek-cell DNA sample and UV transilluminator.
Part B: 1.5ml Microfuge tube, 15ml centrifuge tube capped and racks, micropipettes and tips, 4M Sodium Chloride (NaCl), 10% SDS, cold 70% and 95% Ethanol (EtOH), TE buffer, paper towel and cotton swab, TAE buffer.

Methods:
Part A: To extract DNA from human cells. Labeled 50ml centrifuge tube capped with 50ml of 0.9% NaCl (Saline), labeled two 1.5ml microfuge tubes with name and tube #1 and tube #3. Pipetted 50ml of 0.9% NaCl (Saline) to a 50ml centrifuge tube, then transferred 10ml of saline to a new paper cup, swish the saline inside the mouth for 30 seconds to rinse off cells lining to the saline solution, spit the saline from the mouth to a paper cup and transferred 1ml of the cell-saline liquid into microfuge tube #1 labeled and tighten tube cap, then placed tube #1 to a balanced centrifuge for high speed 1 minute. Poured the supernatant from tube #1 out and added 30μl of 0.9% saline solution to resuspend the cells pellet in tube #1, made sure all the cells pellet mixed with saline solution, then pipetted 30μl of the cells suspension from tube #1 to tube #2 that containing 200μl of 5% Chelex beads and put tube #2 to vortex to mix well. Placed tube #2 with a locking cap and float boat in water bath incubator at 99°C for 10 minutes, and then quickly transferred to ice bath for 1 minute. Put tube #2 to vortex, then placed in the balanced centrifuge for high speed 1 minute. Used micropipette with a new tip to extract 60μl of Cheek-cell DNA sample without Chelex beads to the new tube #3 labeled. Stored the Cheek-cell DNA sample tube #3 in refrigerator at 4°C temperature (long-term storage at -20°C temperature). Run gel electrophoresis to detect the presence of DNA using the same steps in experiment #10 methods section.
Part B: Labeled a 15ml centrifuge tube (tube #1) and a 1.5ml microcfuge tube (tube #2) with date and initial. Pipetted 0.5ml of 4M NaCl to tube #1, used a cotton swab into the mouth to collect cells sample, then submerged the cotton swab in tube #1 that containing 0.5ml of 4M NaCl, added 0.5ml of 10% SDS and mixed gently (did not shake). Used micropipette with a new tip to transfer 0.5ml of cold 95% EtOH to a microfuge tube #2 labeled and 1ml of cold 95% EtOH to centrifuge tube #1. Made sure the cloudy white was off from cotton swab in tube #1, then discarded a cotton swab and slowly pipetted out 0.7ml of cloudy white liquid from tube #1 to tube #2 that containing 0.5ml of cold 95% EtOH. Closed tube #2 cap tightly and put in balanced centrifuge for 5 minutes. Discarded the supernatant from tube #2 and used new pipette tip to add 0.7ml of cold 70% EtOH to tube #2 and put it in the balanced centrifuge for 5 minutes, then discarded the supernatant and used new pipette tip  to add 0.7ml of cold 70% EtOH to tube #2 and discarded ethanol. Dabbed tube #2 lip to paper towel and allowed to dry the DNA completely, then resuspended the pellet with 50ml TE buffer (amount depends on the size of the pellet) to tube #2, made sure DNA off the wall of tube and dissolved with TE buffer and stored in refrigerator at 4°C. Run gel electrophoresis to detect the presence of DNA using the same steps in experiment #10 methods section.

Results/Calculations:
Part A: To prepare 50ml of 0.9% NaCl solution used a formula % Mass/volume:
                    0.9g   x 50ml = 0.45g of NaCl
                   100ml
             To prepare to make 60ml of 0.8 % agarose:
                    0.8g   x  60ml = 0.48g of agarose
                   100ml
Part B: To prepare to make 50ml of 0.8% agarose:
                    0.8g   x   50ml = 0.4g of agarose
                   100ml