1. Basic Instrumentation Handling the instruments form the basis of the practical knowledge and learning its mechanism of working ensures the proper handling and significance of its usage  Related LOs: Environment and health safety issues > Prior Viewing - IDD-1. Extraction of bacterial protein, IDD-6. Extraction of serum protein > Future Viewing – IDD-11. Protein quantification  Course Name: Basic Instrumentation  Level(UG/PG): UG  Author(s): Dinesh Raghu, Vinayak Pachapur  Mentor: Dr. Sanjeeva Srivastava *The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license

2. Learning objectives After interacting with this Learning Object, the learner will be able to: 1. Operate the steps involved in the instruments 2. Analyse the theory and the mechanism of working for different instruments 3. Assess the troubleshooting steps involved in the experiments. 5 3 2 4 1

3. Master Layout 5 3 2 4 1 Colorimetry Centrifugation UV- Spectrophotometric analysis Laminar air flow Slide 4- 12 Slide 13-17 Slide 18-21 Slide 22-24

4. Step 1: 5 2 1 4 3 Scroll Opening for cuvette display T1: Colorimetry Video File: Colorimeters.mp4

5. Step 2: 5 2 1 4 3 T1: Colorimetry Audio Narration Description of the action Absorbance of a sample that is nothing but logarithm ratio of incident light to reflected light is equal to the product of path length, epsilon constant and concentration of the solution. Form the above equation one can calculate the concentration of the solution given the incident light, reflected light and path length value. Animate the above display, like incident light (Io) passing through the sample of concentration ‘c’, travelling a path length “l” and coming out as reflected light (I). Now animate putting these parameters in the equation above for user click to explain accordingly.

6. Step 3: Audio Narration Description of the action 5 2 1 4 3 Show a instrument labeled as “colorimeter” and draw it as shown in the figure. Animate a scroll, a opening and a display screen, auto zero and absorbance buttons. Instruct the user to click on start in the instrument and the user should move the scroll so that the wavelength is set to 570nm as shown in the image and allow it stand for 30 minutes. Use of colorimetry is explained with the steps from IDD-47 quantitative and qualitative estimation of amino acid- ninhydrin, for more information. The instrument need to set to the required wavelength at first place before taking the reading. Once the instrument is set for the wavelength, keep it on stand for 30min to attain the set wavelength. Meanwhile prepare the dilution of samples. T1: Colorimetry

7. Step 4: 5 2 1 4 3 1 4 3 2 T1: Colorimetry Video File: Colorimeters.mp4

8. Step 5: Audio Narration Description of the action 5 2 1 4 3 After 30minutes, instruct user to rinse the cuvette with water. let user set the pipette to 1000ul to take out blank solution to add into the cuvette and show like the user inverting the cuvette and pouring out the solution into a beaker. Instruct the user to take 2000ul of the blank and add to the cuvette, (repeat the above step twice), let user clean the cuvette with tissue, place it in the opening and click on “Absorbance” the reading in the display should show ”0.00”. now ask user to click “auto zero“ option and the display should show “0.00” reading then animate like removing the cuvette and pouring the solution out. Fill the cuvette with the blank solution and take the OD, auto zero the instrument and take the readings for all the sample tubes. T1: Colorimetry

9. Step 6: Audio Narration Description of the action )‏)‏ 5 2 1 4 3 Instruct the user clean the cuvette with blank. Let user fill the cuvette with the solution from the tube labeled as “0.2”. Let user take out the full volume from the tube with help of pipette and trasfer it to cuvette, clean the edges of the cuvette with tissue. Keep it in the opening as instructed and press” absorbance” and note down the readings, remove the cuvette, pour the solution out and follow the same for the solutions in tubes “0.4,0.6,0.8,1,unknown 1,2,3. Show the values as in next slides and the graph T1: Colorimetry Before taking reading for each sample the cuvette need to be rinsed with blank.

10. Step 7: 5 2 1 4 3 SampleOD at 570nm Blank0.00 0.20.01 0.40.03 0.60.05 0.80.07 1.00.09 Unknown 10.02 Unknown 20.04 Unknown 30.06 T1: Colorimetry

11. Step 8: 5 2 1 4 3 T1: Colorimetry

12. Step 9: Audio Narration Description of the action 5 2 1 4 3 Instruct the user to plot the graph as OD in y axis and the concentration in x axis and show like a straight line is drawn (red line) once this is done, animate like the user locating the point on y axis for “unknown 1 (0.02) and drawing a line towards the red line and when the blue line touches the red line drag the blue line down to find the concentration as 0.3mg follow the same for other two unknowns and show the concentration as 0.5mg, 0.7 mg Plot the graph between OD at 570nm and the concentration of the sample and extrapolate the unknown OD value to find the concentration. For colorimetry to work a reaction must be found which will produce a color that can be measured. The absorbing molecules must be uniformed distributed throughout solution. Choice of the wavelength is very important to get the better sensitivity and selectivity. T1: Colorimetry

13. Step 1: T2: centrifugation 5 2 1 4 3 Centrifugerotor Video File: Centrifuge.MTS and Centrifuge_part2.MTSc

14. Step 1: Audio Narration Description of the action 5 2 1 4 3 The animator should draw a centrifuge instrument as shown in the figure. Please include the buttons like enter, set, start, open a regulator nob along with display on the centrifuge. Let user click on “OPEN” button, animate the centrifuge lid opening on its own, let user rotate in anti-clockwise to open the lid of the rotor. Let user keep the lid on table, take out the tubes to centrifuged and place them in the opening. Now balance the tubes in the rotor. Now let user pick the rotor lid and close it by making clock-wise movement. Let user take the centrifuge lid to its normal position and press to close it. Now let user set the required parameters for speed, time and temperature by regulating the nob. Once the parameters are set, let user click on “START” button, animate increase in speed from zero to the set point, along with temperature and count down time display. Transfer the content into the centrifuge tube and perform a centrifugation at required speed, time and temperature. Organelles separate when the density of the organelle equals the density of the sucrose gradient T2: centrifugation

15. Step 2: 5 2 1 4 3 r T2: centrifugation A B http://www.youtube.com/watch?v=IhJNFGfsUus

16. Step : 5 2 1 4 3 1) sedimentation coefficient S = 1/ w^2 r r × dr/dt where w = angular velocity of the rotor in radians/sec r = the distance between the particle and the centre of rotation (m) dr/dt = the rate of movement of the particle (m/sec) 2)RCF g = (1.118 × 10 ^-5 ) R S^2 g- relative centrifugal force, R -radius of the rotor in meters, and S - speed of the centrifuge in revolutions per minute (RPM) 3) Svedberg: Sedimentation rate: dr/dt = w^2r.S T2: centrifugation

17. Step : Audio Narration Description of the action 5 2 1 4 3 Instruct user to carry out centrifugation step. The animator should draw a centrifuge as shown in the figure. Now once centrifugation is going the animator must zoom in the instrument and show the rotor and tube rotating. Meanwhile show the different color moving down and stopping at one point as in the previous slide. And show the formulas and the governing relations in the animation along with the audio narration Centrifugation works on the basis of the centrifugal force which acts away from the center. Relative centrifugal force takes the gravity into account during separation. This force along with the particle density and liquid density helps in the separation of the particles. Particle with high density will sediment faster to precipitate than the low density ones which are left out as supernatant (figure: A). If the particles are of varying density, than different layers are formed after centrifugation (figure: B). The sedimentation rate is expressed in terms of svedberg units T2: centrifugation

18. Step 1: )‏)‏ 5 2 1 4 3 Lid that can be opened Display Options like number 0-9, set wavelength, autozero, absorbance T3:: UV-Visible spectrophotometer Video File: UV vis spectroscopy.flv and UV Spectrometer.MTS

19. Step 1: Audio Narration ‏ Description of the action 5 2 1 4 3 Animate the instrument as in figure and redraw the instruments with the specification mentioned in the figure and zoom the instrument and show a schematic as shown in the figure with the labeling but redraw completely. Go through the IDD enzyme assay for more information. UV-Visible spectrophotometer has a monochromator, light source and sample holder and detector, Light from the source are converted to a monochromatic light of particular wavelength and allow it pass through the sample and amount of light that emerges is detected by a detector. The wavelength used in the specific for the sample to be measured. T3:: UV-Visible spectrophotometer

20. Step 2: 5 2 1 4 3 L T3:: UV-Visible spectrophotometer

21. Step 3: Audio Narration Description of the action 5 2 1 4 3 Now show the figure as in slide (redraw) followed by the formula which is given in the slide, audio narration should take place simultaneously show the animation of air flow in the direction shown in the figure UV-Visible spectrophotometer works on the basis of Beer-Lambert’s law, the law relates the absorbance and the concentration of the solution. In the equation L signifies the path length, C concentration, E (epsilon) absorption coefficient and Io and I corresponds to the intensity of light before entering the solution and the intensity after coming out of the solution. The intensity of the light coming out of the cuvette decreases when the concentration of the substances in the cuvette increases. T3:: UV-Visible spectrophotometer

22. Step 1: 5 2 1 4 3 T4: Laminar air flow CLASS-2 cabinet Video File: Laminar air flow_1

23. Step2 : Audio Narration Description of the action 5 2 1 4 3 Go through the IDD for the bacterial extraction to know the usage of laminar air flow (slide 6,7), let the user click on the laminar flow in the figure to know the mechanism of working of laminar flow and the types of cabinet Laminar air hood is used to maintain the aseptic condition that can be used for microbiological activities. The laminar air flow used in laboratories uses horizontal air flow type with the air flow direction is facing towards the user and the velocity of air flow is maintained constant. The filters used are of different types depending on the type of sample used. The biosafety cabinets is of 3 classes. Class 1- for has HEPA filters that removes contaminants from the exhaust air. This is only for environment protection Class 2- for common usage in microbiological activities, this cabinet has HEPA filters for filtering the entering air and the exhaust air and provide personnel, environmental and product protection. Class 3- for handling most pathogenic microbes especially in maximum containment labs. HEPA (High efficiency particulate air removes 99.97% of particles of size 0.3micro meters. The UV in the cabinet is used prior to the usage of cabinet to kill existing organism in cabinet. Air flow prevents the entry of any microbes from the environment. T4: Laminar air flow

24. Step2 : Audio Narration Description of the action 5 2 1 4 3 Animate to show the flow of air, zoom in the HEPA filter, blocking the microbes. Let user “ON” UV light for 5min,later OFF the UV light. Now let user On the “LIGHT” and ON the “AIR FLOW” and animate user doing the experiment on the laminar work bench. HEPA (High efficiency particulate air removes 99.97% of particles of size 0.3micro meters. The UV in the cabinet is used prior to the usage of cabinet to kill existing organism in cabinet. Air flow prevents the entry of any microbes from the environment. T4: Laminar air flow

25. Animation area Instructions/ Working area Credits Name of the section/stage Interactivity area Tab 02Tab 03Tab 04Tab 05Tab 06Tab 07 Button 01 Button 02 Button 03 Tab01 Slide 4- 12 Slide 13-17 Slide 18-21 Slide 22-24 Interaction1: Animator should frame a question “ if you require a aseptic condition for working with bacteria which instrument you will prefer” a)Centrifuge b)Clean room c)Laminar air flow d)UV-Visible spectrometry Instruction: if the user selects “ laminar air flow” show the animation involving laminar air flow Interaction 2: In slide-17: give user un-know/any sample and instruct to find the exact wavelength for the sample determination? Instruction: let user start taking the absorbance of the sample at all the wavelength, compare the readings and conclude the wavelength for higher absorbance.

26. Questionnaire: APPENDIX 1 Question 1 Absorbance can be taken using a)Calorimetry b)Spectroscopy c)spectrophotometry d)Refractometry Question 2 UV-Visible spectrophotometer works based on a)Beers law b)Lamberts Law c)Beer-Lamberts Law d)Raman spectrum Question 3: As the absorbance increases, the intensity of the outcoming light a)Decreases b)Increases c)Remains same d)zero

27. Links for further reading Reference websites: 1. http://www.stanford.edu/dept/EHS/prod/researchlab/bio/docs/typ es_biosafety_cabinets.pdf 2. http://www.nuaire.com/download/brochure/airegard_laminar_airfl ow_products.pdf 3. http://www.structuralchemistry.org/teaching/downloads/scm08_1 0.pdf 4. http://www.fondriest.com/pdf/thermo_colorimeter_theory.pdf 5. Video for pipette: http://www.labtricks.com/2010/01/11/how-to- use-a-pipette/ 6. Video for buffer preparation: http://www.labtricks.com/2010/01/01/how-to-make-and-ph- buffers/ APPENDIX 2

28. Summary APPENDIX 3 The instruments discussed are routinely used in the proteomics experiment. Each and every step of the instrument as a principle behind, which when followed properly will yield better result.