MEDIA PREPARATION

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Materials Required :

1. LB broth mix
2. LB Agar
3. Distilled water
4. Plates and conical flasks

Procedure :

1. Mix 25g of LB broth powder in 1L of Distilled water.
2. Mix 40 g of LB agar in 1L of distilled water.
3. Autoclave and cool.
4. Pour the broth into a conical flask.
5. Pour the agar mixture onto a plate and let it set.

SUBCULTURE

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INOCULATION

Materials Required :

1. Autoclaved LB broth
2. Pipette tips

Procedure :

1. Pipette out bacterial cells and inoculate into LB broth.
2. Close the conical flask with non absorbent cotton.
3. Incubate the flask in an incubator at 37^oC.

SPREAD PLATE

Materials Required :

1. Dry agar plate (with/without antibiotics)
2. Sterile spreader
3. Parafilm tape
4. Transformed cells in LB broth grown for 1 hour at 37^oC in the shaker.

Procedure :

1. Pipette out of transformed cells in LB Broth onto the center of the surface of an agar plate.
2. Use a sterile plastic spreader.
3. Spread the sample evenly over the surface of agar using the spreader.
4. Wrap the Petri dish with parafilm.
5. Incubate the plate at 37°C for 8-12 hours.

MINI PREP

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Materials Required :

1. P1 - Resuspension buffer- 50mM Tris•HCl (pH = 8), 10mM EDTA, 100μg/ml RNase A
2. P2 - Lysis buffer - 200mM NaOH, 1% SDS
3. P3 - Precipitation buffer - 3M potassium acetate (pH = 5.5)

Procedure :

1. Grow 2 to 6ml culture O/N
2. Transfer 2ml (at a time) to 2ml microcentrifuge tube(s) and pellet at 14000 rpm for 5 minutes. Discard supernatant and repeat as necessary
3. Resuspend in 300μl ice-cold P1
4. Add 300μl P2, gently invert 3x. Quickly add 300μl P3 and invert 3x
5. Spin at 14000 rpm for 10 minutes at room temperature
6. Retain supernatant in 1.5ml microcentrifuge tube
7. Add 650μl of 100% isopropanol, gently invert, and incubate for 10 minutes at room temperature
8. Spin at 14000 rpm for 10 minutes, then discard supernatant
9. Wash pellet with 500μl of cold 70% ethanol
10. Spin at 14000 rpm for 5 minutes
11. Discard the supernatant. Carefully tap the tube to remove remaining ethanol. Dry pellet in vacuum.
12. Resuspend the pellet in 20μl of sterile ddH₂O or TE buffer. Nanodrop to determine concentration. Store at -20°C

3A Assembly

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Materials Required :

1. Two Parts to be joined A and B: Miniprepped
2. Linearized Plasmid Backbone
3. Restriction enzyme: EcoRI, XbaI, SpeI, PstI, DpnI
4. Buffer (10X buffer-50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9 at 25°C.)
5. BSA
6. NFW

Procedure :

1. Enzyme Master Mix for Plasmid Backbone
  a. 5 ul Buffer
  b. 0.5 ul BSA
  c. 0.5 ul EcoRI-HF
  d. 0.5 ul PstI
  e. 0.5 ul DpnI
  f. 18 ul NFW
2. Enzyme Master Mix for Part A
  a. 5 ul Buffer
  b. 0.5 ul BSA
  c. 0.5 ul EcoRI-HF
  d. 0.5 ul SpeI
  e. 18.5 ul NFW
3. Enzyme Master Mix for Part B
  a. 5 ul Buffer
  b. 0.5 ul BSA
  c. 0.5 ul XbaI
  d. 0.5 ul PstI
  e. 18.5 ul NFW
4. Digest Plasmid Backbone by adding 4 ul linearized plasmid backbone (25ng/ul for 100ng total) and 4 ul of Enzyme Master Mix for plasmid at 37^oC for 30 mins.
5. Digest Part A by adding 4 ul Part A (25ng/ul for 100ng total) and 4 ul of Enzyme Master Mix for part A at 37^oC for 30 mins.
6. Digest Part B by adding 4 ul Part B (25ng/ul for 100ng total) and 4 ul of Enzyme Master Mix for part B at 37^oC. 7. Add 2ul of digested Plasmid Backbone (25 ng)
8. Add equimolar amount of Part A (EcoRI-HF SpeI digested) fragment (< 3 ul)
9. Add equimolar amount of Part B (XbaI PstI digested fragment) (< 3 ul)
10. Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
11. Add 0.5 ul T4 DNA ligase
12. Add NFW upto 10 ul
13. Ligate at 16^o C for 30 mins.
14. Transform with 1-2 ul of product

PCR

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Procedure :

1. PCR Setup

2. Temperature Profile

Agarose Gel Electrophoresis

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Materials Required :

1. TAE (EDTA, Acetate, Tris-base)
2. Agarose
3. Ethidium bromide
4. Casting tray
5. Well combs
6. Voltage source
7. Gel box
8. UV light source
9. Microwave

Procedure :

1. Make 1% Agarose Gel. (Mix 1g of Agarose powder with 100mL of 1X TAE buffer)
2. Microwave for 1-3 mins until the agarose is completely dissolved.
3. Let the agarose solution cool down.
4. Add ethidium bromide (EtBr) to the agarose solution of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
5. Pour the agarose into a gel tray with the well comb in place.
6. Let the agarose solution set.
7. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
8. Fill the gel box with 1xTAE (or TBE) until the gel is covered.
9. Add loading buffer to each of your DNA samples.(Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample).
10. Carefully load a molecular weight ladder into the first lane of the gel.
11. Carefully load your samples into the additional wells of the gel.
12. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
13. Using any device that has UV light, visualize your DNA fragments.
14. Using the DNA ladder in the first lane as a guide , you can infer the size of the DNA in your sample lanes.

Gel Extraction

Procedure :

1. Once you have run your gel, move it to an open UV box, remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel.
2. Place the gel in a labeled microfuge tube.
3. Using a scale, weigh the gel fragment because it is directly proportional to its liquid volume and this is used to determine how much of each buffer to add during the DNA isolation step.
4. Add 200 μL Binding Buffer (NTI) per 100 mg gel.
5. Incubate the mixture at 50ºC for 5-10 minutes or until the gel has completely melted.
6. Add the above mix to a EZ column and let it sit for 2 mins, and centrifuge at 11,000 x g for 30s at room temperature.
7. Discard liquid and add 750 μL of wash buffer and centrifuge at 10000 rpm for 2min.
8. Repeat the step twice and discard the flow-through liquid and re-use the collection tube.
9. Place the EZ column into a clean 1.5 mL eppendorf tube. Add 15-30 μL of Elution Buffer (10 mM Tris-HCl, pH 8.5) directly onto the column matrix and incubate at room temperature for 1 minute. Centrifuge for 1 minute at 11,000 x g to elute DNA.

Restriction digestion

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Procedure :

1. Setup

2. Incubate at 37^o C for 2hours.
3. Run Gel electrophoresis

Ligation

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Procedure :

1. Setup

2. The ligated product is in 10 ul aliquot.

Competent Cell Preparation

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Materials Required :

1. LB miller broth
2. E coli cells
3. Conical flasks
4. Falcon tubes
5. Ice and ice box
6. CaCl2 solution (20mM and 0.1M)
7. 80mM MgCl₂ solution
8. 1.5mL microcentrifuge tubes

Procedure :

Chemical Method :

1. Take 10 mL LB broth and inoculate it with E. coli and let it grow for 12 hours. Check OD600.
2. Transfer the colony in 50mL LB miller broth in 250mL (1:5 ratio) conical flask.
3. Now take OD after 30-60 mins until it reaches 0.35-0.4.
4. Transfer culture to sterile ice-cold falcon tubes. Cool culture to 0-4^oC by incubating it on ice for 10 minutes.
5. Centrifuge the culture at 4^o C at 4100rpm for 10 minutes.
6. Decant the medium and resuspend the cell pellet in 30mL of ice-cold MgCl₂ and CaCl₂ solution (80mM MgCl₂ and 20mM CaCl₂) by gentle swirling.
7. Again centrifuge at 4100rpm for 10 minutes at 4^oC.
8. Decant the media and resuspend in 2mL of ice-cold 0.1M CaCl₂ for each culture.
9. Make aliquots of 100 ul each and store at -80^oC.

Transformation

Materials Required :

1. Competent E coli cells
2. Ligated products
3. 1.5 mL microcentrifuge tubes
4. Ice and icebox
5. LB miller broth
6. LB agar with appropriate antibiotic plate (Chloramphenicol)
7. Micropipette and tips

Procedure :

1. Mix 10 ul of ligated products with 100 ul aliquots of competent cells in 1.5mL centrifuge tubes.
2. Leave on ice for 15-30 minutes.
3. Heat shock the mixture in a water bath for 45 seconds at 42^o C.
4. Keep in ice for 5 minutes.
5. Add 1 ul of LB broth (without antibiotics) and let it grow in the shaking incubator for 45-60 minutes.
6. Spread 50 ul on the Chloramphenicol antibiotic plate and place it in a 37^o C static incubator.
7. For the remaining solution, centrifuge at 4000 rpm for 4 minutes and resuspend in 50 ul LB broth in case you want to spread all transform cells or either store the remaining solution at -80^o C.
8. Usually, colonies appear after 12-13 hours.

BIURET TEST

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Materials Required :

1. 1% alanine and 5% albumin or egg white (as positive control)
2. Biuret reagents(Copper sulfate (CuSO₄), sodium hydroxide (NaOH) and sodium-potassium tartrate).
3. Deionised water (as negative control)
4. Dry test tubes
5. Water bath
6. Pipettes

Procedure :

1. Biuret reagent is prepared by dissolving copper sulfate and sodium,potassium tartrate in sodium hydroxide solution. To this solution potassium iodide is added and the volume is made up to 1L with sodium hydroxide.
2. Working standard - it is prepared by mixing protein (egg albumin) with distilled water.
3. To test tubes add 0.5 - 2.5 ml of working standard solution and make it up to 3 ml with distilled water.
4. Then 4 ml of biuret solution is added to all the test tubes.
5. The test tubes are mixed well and incubated at room temperature for 10 mins.
6. Similar procedure is followed for test solutions (unknown solution).
7. The purple colour development is measured at 520 nm.
8. Plot a graph between the protein concentration and optical density. This helps in identifying the protein concentration in the test solution (unknown concentration).

pNPP Phosphatase Assay

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To check the activity of the phosphatase enzyme.

Procedure

Materials Required :

1. Assay Buffer-neutral
2. Assay Buffer-acidic
3. Cell Lysis Buffer
4. Stop Buffer
5. Enzyme / Sample (1 Unit/mL) (Reconstitute enzyme with 1 mL of assay buffer)
6. pNpp Substrate (5 µl of substrate per 96-well)
7. Microplate reader
8. 96-well plates with clear flat-bottom
9. Dulbecco's Phosphate-Buffered Saline (PBS)

Sample Preparation :

1. Gently aspirate the cell culture medium.
2. Wash the cells twice with Dulbecco's Phosphate-Buffered Saline(PBS).
3. Lyse the cells with appropriate amount of cell lysis buffer (0.5 ml for 35 mm dish).
4. Centrifuge the cell lysate at 14,000rpm for 5 min at 4^o C. Transfer the supernatant to a new tube.
5. Perform protein assay to determine total protein concentration in the lysate.
6. We recommend performing several dilutions in the assay buffer to ensure your readings fall within the standard range.

Assay procedure :

1. Apply 45 µl of each enzyme in duplicate to each of a 96-well plate.
2. Add 5 µl of substrate per well and incubate in the dark for 15-60 min.
3. Stop the reaction by adding 50 µl of stop buffer to each well. Mix and measure the absorbance on ELISA plate reader with a test wavelength at 405 nm and a reference wavelength at 630 nm. Subtract the 630 nm reference absorbance from the 405 nm measurement.

BIOFILM FORMATION ON DIFFERENT SURFACES

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Materials Required :

1. E.coli stock culture.
2. LB-MES broth.
3. 96 well plate.
4. Thermostable static incubator.
5. Orbital shaker.

On Plastic Biocarriers.

Procedure :

1. The initial E.coli stock culture was prepared by adding 2 ml inoculum into 20 ml standard LB-MES broth with a pH of 5.5 and allowing the E.coli to grow overnight (16-18 hrs) at 28^oC under orbital shaker at 120 rpm.
2. Next day, the optical density (OD) at 600nm was checked. Value at 600nm must be 2.0 or dilute it.
3. The culture was diluted in the ratio of 1:100 with 20 ml of fresh LB - MES broth at a pH of 7.4 and 0.5 ml of culture in a conical flask.
4. Then add GAC (0.4-0.5 g/cc) or PE plastic (0.94-0.96 g/cm3) bio carriers into the conical flask.
5. After 48 hrs the biocarries are removed and sent for SEM analysis.

Congo Red Assay

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To visually see the production of curli by bacteria.

Materials Required :

1. LB broth
2. Congo Red stain
3. Chloramphenicol antibiotic(2mg/ml)

Procedure :

1. Add bacteria in 5mL of culture media
2. Add 50µL of the appropriate selective antibiotic
3. Let bacteria grow at 30°C with a 180 rpm agitation until OD600 = 1
4. Take 5mL of the bacterial culture in a 15mL tubes
5. Pellet down the cells at 13000 rpm for 5 mins
6. Remove the supernatant
7. Add 3mL of the Congo Red solution
8. Let incubate for 10 mins
9. Pellet down the cells at 13000 rpm for 5 mins
10. Remove the supernatant
11. Compare the results with the positive and negative control.

Biofilm Assay Using Safranin

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To perform quantification of biofilm formation

Materials Required :

1. 96 well plate with clear flat-bottom
2. Overnight cultures
3. 0.1% safranin stain
4. Ethanol
5. 30% acetic acid

Procedure :

1. Inoculate a 5ml liquid medium with 5μl 1st Overnight culture, use disposable test tubes and incubate at proper conditions overnight.
2. Add the 2nd overnight culture onto plates and perform the experiment as follows.
3. Add 200μl of diluted 2nd overnight culture into each well (96 well plates) and incubate at proper conditions overnight.
4. Discard the overnight bacteria and wash with Ethanol.
5. Add 200μl 0.1% safranin per well to stain the biofilm.
6. Wait for a couple of minutes, then discard unabsorbed safranin and wash once with ethanol.
7. Add 200 μl of 30% acetic acid into each well and wait for 5 minutes.
8. Measure absorbance of samples at OD of 530 nm.
9. Carefully discard the plate in the biological waste bin after measurement of OD.

HRP Assay

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Materials Required :

1. 96-well microplates
2. TMB substrate - 3-3'-5-5'-tetramethylbenzidine
3. Horseradish peroxidase.
4. Stop solution (0.16M sulfuric acid)
5. ELISA plate reader.

Procedure :

1. Add 100 µL of TMB substrate to each well containing horseradish peroxidase reaction.
2. Incubate at room temperature for 15 to 30 minutes.
3. Add 100 µL of stop solution (0.16M sulfuric acid)to each well.
4. Measure absorbance at 450nm.

Biofilm Assay Using Crystal Violet

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Experimental Procedure :

The experiments were performed on a 96 well plate.

1. The initial E.coli stock culture was prepared by adding 1000 ul inoculum into 9000 ul standard LB-MES broth with a pH of 7.4 & 5.5 and allowing the E.coli to grow overnight (16-18 hrs) at 37^oC and 28^oC respectively under orbital shaker at 120 rpm.
2. Next day, the optical density (OD) at 600nm was checked. Value at 600nm must be 2.0 or dilute it.
3. Few wells of plate are filled with 200 ul of fresh LB as blank.
4. The culture was diluted in the ratio of 1:100 with 195 ul of fresh LB - MES broth at a pH of 7.4 and 5ul of culture in the well.
5. The plates were then transferred to thermostatic static incubators maintained at 25℃.
6. To avoid evaporation, the external area of the plate was filled with less amount of distilled water.
7. After 24 hrs, the 96 well plate was taken from the incubator and the planktonic cells were removed.
8. The wells were then washed using sterile PBS.
9. Each well was then added with 200 ul of 0.1% crystal violet and was kept for 15 mins for staining.
10. The crystal violet was then removed and washed with sterile PBS and kept the plate for air drying for 5 mins.
11. The stained biofilm was then solubilised by adding 200 ul of 30% acetic acid.
12. The plate was then kept in a microplate reader and OD value was recorded at 570 nm.

Cadmium Toxicity Test

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Experimental Procedure :

1. E.coli cells were grown in LB - MES overnight (14 - 16 hrs).
2. Next day, 1% culture suspension was added to freshly prepared LB - MES and incubated at 37^oC until OD (600nm) reached 0.5.
3. Then sterile CdCl₂ solution was added to various conical flasks with different concentrations (0.18mg/ml),(0.02mg/ml),(0.01mg/ml), all the flasks were then placed in an orbital shaker incubator at 37^oC and 130 rpm.
4. Samples were taken at an interval of 30 mins and OD at 600nm was taken spectrophotometrically.
5. Simultaneously viable cell count was done on LB agar from which cell survival was determined.
6. OD vs conc graph is plotted.
7. For checking the morphology of the cells, the cells are subjected to gram staining and are observed under the microscope.

MTT Assay

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Materials Required :

1. Cell Suspension
2. 96 well plates
3. ELISA reader
4. Hemocytometer

Procedure :

Day 1

1. Cells are grown overnight.
2. Cells grown in suspension are harvested by centrifugation at 500 rpm for 5 mins at 2-8^o C.
3. Discard the supernatant and resuspend the pellet in 5 ml of sterile PBS. Again centrifuge at 500 rpm for 5 minutes at 2-8^o C.
4. Resuspend the pellet in 1ml of PBS.
5. Count the number of cells using hemocytometer and record.
6. Using the sterile PBS serial dilute the cells to 2 fold.
7. Plate the cells at 100 μL per well. Incubate the cells for 6-12 hours at 37^o C.

Day 2

1. Add 10 μL of the MTT solution to each well. The Blank contains 10 μL of MTT solution and 100 μL of PBS.
2. Then incubate the plate for 2-4 hours at 37^o C.
3. View the cells periodically for the appearance of purple precipitate (intracellular precipitate) using an inverted microscope.
4. Add 100 μL of the solubilization solution to each well and mix thoroughly using a pipette.
5. Leave the plate covered in the dark at 37^o C for at least 2 hours. Samples are read after 2 hours.
6. Remove the plate cover and measure the absorbance of the wells, including the blank, at 570 nm.

Sterilization Methods

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1. Autoclave

Autoclaving is a process by which high pressure steam is used to clean and sterilize lab equipment and solutions, as well as breakdown nutrient rich tissues that could become biohazard. The steam, which is kept at 121°C, is far above the boiling point of water and is consequently fatal to most microbes and viruses.

Materials Required :

1. Glass ware
2. Autoclave
3. Autoclave tray
4. Heat resistant gloves

Procedure :

1. Before placing the glassware into the autoclave, ensure enough water has been filled till the required level for the autoclave to function.
2. Place your glass wares into the autoclave after carefully packing them.
3. Using the foot pedlar, close the autoclave machine and tightly screw all the valves.
4. Ensure that the pressure outlet is loosened.
5. Keep the temperature at 121^oC for 50 minutes.
6. After completion of autoclave, using the heat resistant gloves, remove the hot glassware from autoclave and place them in the tray.

2. Fumigation

Fumigation is a process where fumigant, a chemical at required temperature and pressure that can exist in a gaseous form in sufficient concentration will be lethal to pest organisms. It is a very useful method to control insect pests, diseases,and nematodes. It is also used for the reduction of microbial agents on surfaces.

Materials Required :

1. Personal Protective Equipment set that includes face protection, goggles and mask or face shield, gloves, gown or coverall, head cover, and rubber boots.
2. Formaldehyde solution (37%)
3. Distilled water (D/W)
4. Potassium permanganate
5. Ammonia

Procedure :

1. Thoroughly clean windows, doors, floor, walls, and all washable equipment with soap and water.
2. Close windows and ventilators tightly. If any openings are found, seal it with cellophane tape or other material.
3. Switch off all lights, A/C, and other electrical electronic items.
4. Calculate the room size in cubic feet and calculate the required amount of formaldehyde as given in.
5. Adequate care must be taken during wearing PPE that includes face protection, goggles and mask or face shield, gloves, gown or coverall, head cover, and rubber boots.
6. Electric boiler fumigation method for every 1000 cubic feet of the volume of the operation theater-500 ml of 37% formaldehyde added in 1000 ml of water in an electric boiler. Switch on the boiler, leave the room and seal the door.