Department of Bio-chemistry (JNMC)

SOPs

Dept. data last updated on :27/11/2021

ELISA Reader

SOPs for ELISA Reader


1. PURPOSE

1.1 To determine the optimal concentration of a relevant capture antibody or soluble antigen.

1.2
To determine the optimal concentration of a lot of specific enzyme linked secondary antibody.

1.3
To determine the suitability for use of a lot of substrate buffer as measured against a lot of known performance.

1.4
To determine the suitability for use of a lot of PNPP substrate as measured against a lot of known performance.



2. SCOPE

2.1
To evaluate reagents to be used in quantitation of 12.8 antibody in solutions.

2.2
This assay can also be used to evaluate reagents for use in most ELISA procedures.


3. RESPONSIBILITY

3.1
All personnel performing optimization of ELISA reagents are responsible for adhering to this procedure.



4. REFERENCE AND APPLICABLE DOCUMENTS

4.1
TM-0060, ELISA for the quantitation of 12.8 antibody in solutions.


5. MATERIALS AND EQUIPMENT

5.1
See section 9.1


6. HEALTH AND SAFETY CONSIDERATIONS

6.1
See company safety manual.



7. DOCUMENTATION REQUIREMENTS

7.1
Laboratory Notebook 7.2 Attachment A. Reagent Qualification Form


8. GENERAL

8.1

New reagents should be qualified prior to use in an ELISA procedure.

8.2
By titering new or unknown reagents together with known reagents in a ELISA based assay a optimal concentration for use can be determined for the new component.

8.3
The suitability for use of new preparations can be determined by comparing new buffers or substrate preparations side by side with old preparations. The new preparations should give results similar or superior to the old lots.

8.4 ELISA plates should be covered during incubations to prevent evaporation and contamination.





9. PROCEDURE

9.1     Materials and Equipment

9.1.1
Microplate reader/recording spectrophotometer capable of reading @ 405 nm +/- 10 nm (Molecular Devices V-Max).

9.1.2  Multi-channelmicropipettes able to dispense up to 200 uL (8 Channel Finn pipette 50-300 ul).

9.1.3
Single channel micropipettes able to dispense up to 200 uL (Gilson 20-200 ul).

9.1.4     200 uL sized tips for micropipettes (Intermountain P-3200-1).

9.1.5     12 x 75 mm borosilicate tubes (VWR 60824-546).


9.1.6
   15 mL conical tubes (Falcon 2095)

9.1.7     50 mL conical tubes (Falcon 2070)

9.1.8     1.0 mL serological pipets (Falcon 7520)

9.1.9     10 mL serological pipets (Falcon 7551)

9.1.10   Reagent troughs or square petri plates for use as reservoirs (Falcon 1003).


9.1.11

Polystyrene flat bottomed 96 well microtiter plate (Nunc maxisorb ELISA plates) 9.1.12    Vortex mixer (Vortex - Genie2) 9.1.13 ELISA plate Washer (Dynatech ultra wash 2)

9.1.14    Disposable sealing tape for 96 well plates (Corning #430454).


9.2       
Reagents

9.2.1     Dulbecco's Phosphate buffered saline (D-PBS without Ca ++ and Mg ++, JRH # 210-3025)

9.2.2   PT buffer = 0.5% tween 20 (Sigma # P-1379) in 1x D-PBS + 0.01% Thimerosal (Sigma T5125)

9.2.3  
PB buffer = 1.0% Bovine serum albumin (Miles Fraction V reagent grade # 210-3025)    In Phosphate buffered saline buffer + 0.01% Thimerosal (Sigma T5125).


9.2.4
   AMP buffer 0.1 M 2-amino-2-mehtyl-1, 3-propanediol buffer, in 0.01% MgCl2 pH 10.3.

9.2.4.1
Dissolve 10.51 gm of 2-amino-2-methyl-1, 3-propanediol (Sigma # A9754) + 100 mg MgCl2 (Sigma M9272) per 1.0 L Deionized or distilled H2O + 0.01% Thimerosal (Sigma T5125). Make 5.0 litres.


9.2.4.2
Assign a lot number to this batch of buffer and assure that the number is written on each bottle along with the date and the initials of the technician preparing the buffer.


9.2.4.3
Each lot of buffer will be compared against the previous lot as described in this procedure. If this is the reagent to be qualified then samples of both the new and old lot will be needed.

9.2.4.4   Store the AMP buffer at 5 oC and make a new lot once/month.

9.2.4.5   Other substrates or buffer systems (e.g., HRPO) can be evaluated using this protocol. Follow instructions in SOP for particular assay for reagent preparations. 9.2.5 PNPP substrate solution - Dissolve 1.0 MG/ML p-Nitrophenyl phosphate (Sigma 104-100 or Zymed # 00-2201) in amino-2-methyl-1,3-propanediol buffer from 5.2.4. Each new lot of powdered PNPP will be qualified according to this procedure by comparing it against a previous lot. If this is the reagent to be qualified then samples of both the new and old lot will be needed. (Store PNPP at -20 ℃ (+/-5 ℃) in dark containers.)

9.2.6    Capture antibody or antigen used for coating plate. If this is the reagent to be qualified then samples of both the new and old lot will be needed.


9.2.7   Alkaline Phosphatase conjugated secondary antibody. If this is the reagent to be qualified then samples of both the new and old lot will be needed.

9.2.8    Bicarbonate buffer - 0.05 M pH 9.6 (Sigma # C-3041) make from powder; store at room temperature for up to 6 months.

9.2.9   Primary antibody or antigen for which the particular assay being evaluated is designed. If this is the reagent to be qualified then samples of both the new and old lot will be needed.

9.3      Evaluation of optimal concentrations of capture antibody (or antigen).

9.3.1
Prepare 1.5 mL of a 20 ug/mL solution of capture antibody or soluble antigen in Bicarbonate buffer.

9.3.2    Add 100 ul of bicarbonate buffer to each well of a 96 well ELISA plate.

9.3.3    Add 100 ul of coating material from 5.3.1 to each well of the first row of the ELISA plate. Titrate this material down the rows of the plate discarding the 100 ul of excessl material remaining from the last row.

9.3.4    Allow plate to incubate at 2-8 oC for 18-22 hr. 9.3.5 Wash the plates 3 times with PT buffer by sequentially dispensing >200 ul of buffer into the plate and aspirating off (repeated a total of three times using the plate washer).

9.3.6    Block the plates by adding 100 ul of PB buffer to each well.

9.3.7   Allow plates to incubate for 1.0 hour at R.T. and wash the plates with PT buffer as follows: 9.3.7.1 Wash 3 times as described in 5.3.5. Fill the plate with PT buffer.

9.3.7.2
Allow the plate to sit at R.T. for 5 minutes.

9.3.7.3
Wash 3 more times as described in 5.3.5. 9.3.8 Add 100 ul of PT buffer to each well of the plate.






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Flame photometer

SOPs for Flame Photometer


    The principle of flame photometer is based on the measurement of the emitted light intensity when a metal is introduced into the flame. The wavelength of the colour gives information about the element and the colour of the flame gives information about the amount of the element present in the sample.

Compounds of the alkali and alkaline earth metals (Group II) dissociate into atoms when introduced into the flame. Some of these atoms further get excited to even higher levels. But these atoms are not stable at higher levels. Therefore, these atoms emit radiations when returning back to the ground state. These radiations generally lie in the visible region of the spectrum. Each of the alkali and alkaline earth metals has a specific wavelength.


Application


     The flame photometer is one of the most useful instruments in clinical analyses. This is due to the suitability of the flame photometer for determining sodium, potassium, and calcium, which are of immense importance in the development of the living being and indispensable to physiological functions. In the clinical analysis of sodium and potassium, the flame photometer gives, rapidly and accurately, numerous differential data for normal and pathological values.


Working Steps


·     Both the standard and sample solution are prepared in fresh distilled water.

·     The flame of the photometer is calibrated by adjusting the air and gas. Then the flame is allowed to stabilize.

·     Then switch on the instrument and the lids of the filter chamber are opened to insert appropriate colour filters.

·     The readings of the galvanometer are adjusted to zero by spraying distilled water into the flame.

·     The sensitivity is adjusted by spraying the most concentrated standard working solution into the flame. Now the full scale deflection of the galvanometer is recorded.

·     Again distilled water is sprayed into the flame to attain constant readings of galvanometer. Then the galvanometer is readjusted to zero.

·     Now each of the standard working solutions is sprayed into the flame for three times and the readings of galvanometer are recorded. After each spray, the apparatus must be thoroughly washed.

·     Finally, sample solution is sprayed into the flame for three times and the readings of galvanometer are recorded. After each spray, the apparatus must be thoroughly washed.

·     Calculate the mean of the galvanometer reading.

·     Plot the graph of concentration against the galvanometer reading to find out the concentration of the element in the sample.

·     The solvent is first aspirated to obtain fine solid particles.

·     These molecules in the solid particles are moved towards the flame to produce gaseous atoms and ions.

·     These ions absorb the energy from the flame get excited to high energy levels from the ground state.

·     But as these ions are unstable, they return back to ground state. While returning they emit characteristic radiation.

·     The intensity of emitted light is proportional to the concentration of the element.



Precautions


  • When using certain substances, the liberation of hazardous gases may require the use of a fume cupboard or other means of extraction.

  • Be aware of biological contamination. Ensure sterilization of the capillary tube, waste, nebuliser and mixing chamber is performed after contamination.

  • Ensure equipment is used on a clean, dry, non-combustible, solid work surface with at least 300mm suitable clearance all around andat least 1m clearance above the chimney.

  • Ensure that the outer chimney is properly installed before operatingthe equipment.

  • Ensure that the gas and air pipes have been connected correctly and that there are no gas leaks present before igniting.

  • Unit should be properly connected to the gas cylinder making sure there are no gas leaks.

  • Take care when fitting and removing the capillary tube to the nebulizer needle.

  • Do not leave the flame photometer unattended during operation.

  • Do not look down the flame chimney when igniting or operating the equipment.

  • Do not handle the flame chimney whilst in operation.

  • Do not lean or stretch over the equipment whilst in operation.

  • Do not cover the chimney whilst in use. Do not block or obstruct ventilation slots.

  • Do not spill substances onto the unit. If spillage does occur, disconnect unit from mains supply. If using hazardous substances

  • Allow sufficient time for the chimney to cool before handling.

  • Over adjustment of the fuel valve will cause excess flame.

  • Avoid installing the flame photometer in areas that are susceptible to drafts, but ensure that there is adequate ventilation to prevent any build-up of gas.

  • In order to completely disconnect power to the flame photometer the mains power cord must be detached from the back of the unit.

  • This hazard symbol is present on the flame chimney outer casing and indicates that the surface may be hot to touch.


Water bath

SOPs for Water bath


A water bath is laboratory equipment made from a container filled with heated water. It is used to incubate samples in water at a constant temperature over a long period of time. Most water baths have a digital or an analogue interface to allow users to set a desired temperature, some have their temperature controlled by a current passing through a reader


SOP (Standard Operating Procedure)


1.     Ensure that the platform is dry.

2.     Connect the power supply.

3.     Ensure the water level in water bath. It should be sufficient to pour the heating element.

4.     Switch “ON” the main power supply and instrument mains.

5.     Set the desired temperature of the water bath by rotating the temperature adjustment knob and indicator bulb shows red light until the set temperature is not achieved.

6.     The temperature sensor will maintain the set temperature during use of water bath.

7.     Switch “OFF” the instrument mains & main power supply after use.

1.     It is used in warming of reagents

2.     Melting of substrates

3.     Incubation of cell cultures.

4.     It is also enables certain chemical reactions to occur at high temperature.


Precautions


Glowing of Red lamp indicate mains “ON” & glowing of yellow lamp indicate heater “ON”








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Incubator

SOPs for Incubator


An incubator is a device used to grow and maintain 
microbiological cultures or cell cultures. The incubator maintains optimal temperaturehumidity and other conditions such as the CO2 and oxygen content of the atmosphere inside. Incubators are essential for much experimental work in cell biologymicrobiology and molecular biology and are used to culture both bacterial and eukaryotic cells.The most commonly used temperature both for bacteria such as the frequently used E. coli as well as for mammalian cells is approximately 37 °C (99 °F), as these organisms grow well under such conditions.


SOP (Standard Operating Procedure)


  1. Ensure that the incubator is properly connected to the power supply.

  2. Switch “ON” the main switch and then the cabinet switch, the indicator will glow green, red andgreen, respectively.

  3. Set the required temperature on digital display by pressing the set button along with arrow button upto increase the temperature/down to decrease the temperature.

  4. Observe the temperature shown on the digital display. The temperatureshould not differ by ±2°C.

  5. After achieving the desired temperature keep the samples inside the incubator on the tray and close the door firmly.

  6. Record the temperature of Incubator.

  7. Report any discrepancy observed during operation or temperature monitoring, to QC Manager.


Uses


Laboratory incubators provide a controlled, contaminant-free environment for safe, reliable work with cell and tissue cultures by regulating conditions such as temperature, humidity, and CO2. Microbiological incubators are used for the growth and storage of bacterial cultures.


Precautions

  1. Doorways and vents can blow in contaminants and increase the chance of fungal growth. What’s more, they can create drafts which can affect the temperature stability of your unit.

  2. Direct sunlight can cause temperature fluctuations and issues with anti-condensation features.

  3. We need enough space (at least three inches) around the unit so that heat can vent and cords and sockets are easily accessible.

  4. Place floor incubators on a stand to limit the risk of contaminants sweeping in when the door is open.

  5. Avoid humid, damp areas that may be harbouring fungal growth.

  6. Place units away from sources of vibration such as shakers, stirrers, or refrigerators, as vibrations can affect cell growth.

  7. Ensure the area around the unit is as clean as possible.


Colorimeter

SOPs for Colorimeter


Colorimetry is the measurement of the wavelength and the intensity of electromagnetic radiation in the visible region of the spectrum. Colorimetry can help find the concentration of substances, since the amount and colour of the light that is absorbed or transmitted depends on properties of the solution, including the concentration of particles in it. A colorimeter is an instrument that compares the amount of light getting through a solution with the amount that can get through a sample of pure solvent. A colorimeter contains a photocell is able to detect the amount of light which passes through the solution under investigation. The more light that hits the photocell, the higher the current it produces, hence showing the absorbance of light. A colorimeter takes 3 wideband readings along the visible spectrum to obtain a rough estimate of a colour sample. Pigments absorb light at different wavelengths.


SOP of Colorimeter


  1. Switch on the Colorimeter.

  2. Wait until the fluctuation will stop.

  3. Set the percentage transmission at zero.

  4. Take the Cuvette and clean it very properly. There should not be any trace on the surface of the Cuvette.

  5. Fill the sample in the Cuvette which we are going to measure.

  6. Place the Cuvette very slowly in the Cuvette chamber.

  7. Note down Optical Density of the given sample.

  8. Switch off the instrument.


Uses

1.      It is used by hospitals as well as laboratories for analysing biochemical samples such as urine, cerebrospinal fluids, plasma, biochemical samples, and serum. 

2.      It is widely used to generate a quantitative estimation of the serum components, proteins, glucose, and various biochemical compounds.

3.      It is also used in food industries and by manufacturing industries to make textiles and paints


Precautions

1.      This instrument is a precision measuring instrument.

2.      This instrument is not waterproof and should not be used in high humidity environment or water mist.

3.      Keep the instrument clean and tidy.

4.      If the instrument will not be used for a long time, remove the battery.

5.      The power adapter other than our company's special one cannot be used, otherwise the instrument may be burnt down.

6.      After the instrument is used, the colorimeter and the whiteboard cover should be placed in the instrument box and stored properly.

7.      The instrument should be stored in a dry, cool environment to avoid damage to the instrument.




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Office of the Department

The following staff members are responsible for smooth functioning of office work.


  • Mr. Syed Faisal Viqar

  • Mrs. Huma Shahid

  • Mr. Mohd. Faizan Ali


  • Receive & Dispatch:

All the paper received in the office by Mrs. Huma Shahid. Details of paper are entered in the receipt register.

Papers are shown to the chairman for his/her comment.

Chairman returns the paper with his comment within three days.

The paper are their placed in the respective file folders (Office file/Personal file/Circular file/ Notice Board file/ PG file/NAAC/IQAC/File for Minutes of BOS).

Papers are then circulated/put on the Notice Board or given to concerned staff Teaching and Non-Teaching.

All papers, which are to be despatched from the office. The details are first entered in the despatch register.


  • Store

The following staff members are responsible for smooth functioning of store Mr. S. Nasir Ali (financial matters and matters related to Purchase).


  • Purchase of the following items are done

- Chemicals

- Equipments

- Departmental items (Fan, A/C, Furniture, Stationary etc).

All purchase/repair of the amount ranging between 1 – 25,000 – are done by Chairperson

Purchase ranging between 25,000 – 2.5 lakhs -  through the purchase Committee.

Purchase >2.5 lakhs are done through Finance office of the University.

  • Purchase of various items are done as follow / through the following processes to ask for quotations (at least four) are first asked by the concerned company/supplier.

The lowest cost quotation is considered, by the approval of Chairman.

Order is placed after Chairman approval.

It should be procured at the earliest possible time.

The received items are entered in the stock register and kept in the store.

Then depending upon the requirements of UG lab / research lab / PG lab / Clinical lab they are issued through the Indent book.


  • Indent book carries the following details:

Name of indenter

Name of items

Quantity


  • Indent book is then approved / permitted  / signed by the Chairman and the items are issued.
  • The following registers are maintained by the store keeper

Register for record of all the instruments in the department.

Register for Chemicals and glass ware.

Register for stationary.

Register for various items of the department

Register for record of repair of various items

Register for day to day expenses.

Miscellaneous

Budget Control Register

MAS Control Register

Register for consumables items

Register for Bank related work TA, DA.

Financial matters of Research Projects operated in the department are also taken care of by store keeper of the department.


  • Maintenance

Person concerned write an application related to the repair work and gives it in the
office of the Department.

Chairperson mark the application to the store incharge for necessary action.

Store incharge calls the person related to that particular work and ask Quotation for rates.

Chairperson  approved quotation of lowest rates.

Repair work is done at the earliest.

SOPs for Department in General


  • Official Working Hours 8:00 AM – 4:00 PM.

  • Research Scholars and MD students work in the research scholars even beyond these hours.

  • The concerned person will collect the keys from Chairman and open the department at 7:45 AM followed by opening of office labs, Chambers of the staff etc.

  • The extra lights switched on at night in the corridors, are switched off in the morning.

  • Sweepers are responsible for cleaning the department and ambience. Indoor plants and water cooler are taken care of by the non-teaching staff and the particular committee constituted.

  • All the staff members and students enter signature in the attendance register, in the displaying “on leave” if any is on leave.

  • Concerned staff update the (board name / designation board) and Notice board. Members of cleanliness and Ambience committee as well as members of the department take care of cleanliness.

  • Before closing the department all, the electrical switches and water taps are checked.

  • All the research scholars and MD students check the  electrical points and water taps before leaving the lab.


COVID-19

  • INSTRUCTIONS

Get Vaccinated

Keep physical distance of at least 1 meter from others.

Avoid crowd and close contact.

Wear a properly fitted  mask

Clean hands frequently with alcohol-based hand rub or soap and water.

Cover mouth and nose with a bent elbow or tissue when cough or sneeze.

Dispose off used tissues immediately and clean hands regularly.


  • Warning Signs for COVID 19

Fever

Dry cough

Headache

Tiredness

Dizziness

Breathlessness

Loss of smell and taste

Diarrhea


  • Seek Medical care immediately if any of these is present
  • If tested positive, isolate until recovery.

Clinical Lab

SOPs for Clinical Lab of the Department

 

v  Surface disinfection: lab bench is cleaned daily with any of the following Spirit- 70% alcohol or antiseptic/ Lysol/ Bleach (0.05%) solution. Preparation of bleach solution: 7gm or half tablespoon full per one litre of water 0.5%- 1:10 dilution; 0.05%- 1:100 dilution; 1% - 1:5 dilution, 0.1% -1:50 dilution)

 

v  The sample collection time is from8:30 am to 12:30 pm on weekdays and 8:30 am to 10:30 am on Friday.

 

v  For the analysis of Liver Function Tests (LFTs) 2 – 3 ml whole blood is required in serum vacutainer.

 

v  After collection, the samples are taken from collection desk to the clinical lab, where they are sorted and labelled.

 

v  Following this the particulars of the samples are entered in the lab register.

 

v  Samples are then centrifuged and serum is separated for performing the tests.

 

v  Haemolysed samples are discarded.

 

v  The tests for Bilirubin (direct and Indirect), AST, ALT, ALP and amylase are performed colorimetrically.

 

v  Calculations are done manually to obtain results.

 

v  The reporting is done following this on the investigation forms and the reports are also entered on the lab register for future reference.

 

v  The reports are available from 3:30 pm onwards from the collection counter itself.

 

v  Reagents are prepared on a weekly basis or as and when required.

 

v  Staff on duty follow the personal protective measures (use of gloves, mask, apron)

 

v  SR’s on duty supervise overall investigations and look for quality control.

v  Bio-medical waste generated is disposed off as per the guidelines

 

   

SOPs for Spill Management

 

1.0         Purpose

The purpose of this document is to provide guidance  during management of  accidental spills.

 

2.0         Scope

This document is use as a guidance document for management of  accidental spills for Clinical Biochemistry Laboratory, Department of Biochemistry, J.N.Medical College, AMU, Aligarh.

 

3.0         Roles and Responsibilities

3.1         Laboratory staff: It is responsibility of Mr. Ashfaq Ai, Mr. Raziuddin & Mr. Arsad Ahmad Laboratory technician to read and follow content of applicable SOP of Spill Management.

 

4.0         Procedure

4.1         Wear disposable gloves.

4.2          Cover spills with absorbent material and pour 1% disinfectant around  the spill and over the absorbent material and leave for 30 minutes.

4.3         And prohibit the place where spillage occurred by keeping a signage board.

4.4         Remove the absorbent material using brush and pan and place in the biohazard bags of infectious waste.

4.5         Wipe off the surface again with disinfectant.

4.6         Sweep broken glass etc. with a brush and collect in a dust pan. Discard into the sharps disposal unit/puncture proof container.

4.7         Report the spills to the laboratory In charge.

4.8         Keep a written record of such accidents.

 

5.0         Appendix

Appendix 1:Spill management log

 

6.0         References

6.1         NACO Manual

6.2         ISO 15189:2012

 

7.0         Appendix 1: Spill Management

 

Date

Spill

Reported By

Cleaned By

Checked by

 

 

 

 

 

 

 

 

 

 

 

 

SOPs for Biomedical Waste Management



8.0         Purpose

The purpose of this document is to provide guidance for proper segregation, treatment and disposal of biomedical waste generated by Clinical Biochemistry Laboratory  in a safe manner.

 

9.0         Scope

This document is used as a guidance document for proper segregation, treatment and disposal of biomedical waste generated by Clinical Biochemistry Laboratory, Department of Biochemistry, J.N. Medical College, AMU, Aligarh.

 

 

10.0     Roles and Responsibilities

10.1     Laboratory staff: It is responsibility of all laboratory staff to segregate the waste generated at the point of generation according to the SOP. 

10.2     Safai Karamchari: It is the responsibility of safai karamchari to take away the segregated biomedical waste daily

10.3     In Charge of Clinical Lab/Chairperson: In Charge of Clinical lab/Chairperson will ensure the compliance of the SOP.

    

10.4     Safety Issues and Waste Handling

10.4.1    Handle all the samples with care , as they can be potentially infectious.

10.4.2    Wear protective laboratory clothing’s in laboratory areas.

10.4.3    Do not pipette any reagent by mouth. Prevent splashing or spilling of samples or solutions containing samples. In case  of  spillage,  immediately  clean  it  with  1%  freshly  prepared  sodium hypochlorite. and dispose of the cleaning material by a suitable method.

10.4.4    Wash hands thoroughly with disinfectant after completion of the test.

 

10.5          Procedure  for segregation, transport and storage of biomedical waste  

10.5.1    Waste segregated at source as soon as it is generated. Refer to Appendix 1

10.5.2    Non-Infectious waste collected in black bags and disposed as general waste.

10.5.3    Infectious plastic is collected in labeled red bags after disinfection, sterilized by autoclaving and sent for disposal

10.5.4    Glassware disinfected by Sodium hypochlorite and sterilized by autoclaving  followed by  cleaning. Never attempt cleaning before Autoclaving.

10.5.5    The liquid infectious waste should be treated with chemical disinfectant for decontamination then neutralized and flushed  into sewer.

 

10.6          Document and Records

10.6.1    Laboratory technician maintain records related to the generation, collection, reception, storage, transportation, of bio-medical waste in accordance with the SOP and any guidelines issued.Refer to Appendix 1.

10.6.2    All records shall be subject to inspection and verification by the prescribed authority at any time.

10.6.3    Date and total number of each bag ,sterilization of Plastic waste and sharps container is recorded in a register and receipt of the same is maintained in the register.(Refer to  Appendix 03 & 04)

 

11.0          Appendix

11.1          Appendix 1: Table of Waste Segregation in color coded bags.

11.2          Appendix 2: Log of Disposal of BMWM Bags/Container.

11.3          Appendix 3: Log of Biomedical Waste Autoclaved

12.0     References

12.1     NACO Manual

12.2     ISO 15189:201

 

APPENDIX 1

Table Of Waste Segregation In Color Coded Bags

Color coding

Waste category

Red

Soiled disposable items like plastic syringes, Vacutainers, serum storage vials, used rapid test devices, used ELISA plates, micro tips; gloves, reagent vials

Yellow

All non sharps and non plastic infectious waste including human anatomical tissues,  cotton, filter paper, gauze contaminated with blood/ body fluids

White Puncture Proof

Needles, syringes with fixed needles, needles from needle tip cutter or burner, scalpels, blades, or any other contaminated sharp object that may cause puncture and cuts. This includes both used, discarded and contaminated metal sharps.

Blue

Broken or discarded and contaminated glass including

medicine vials and ampoules except those contaminated with

cytotoxic wastes. Body Implants

Black

general non infectious waste like paper, wrapper, cardboard box, kit boxes

 




















APPENDIX 2

LOG OF DISPOSAL OF BMWM BAGS/CONTAINER

 

Date

Time

No. of  BMWM Bags/Container

Signature of Safai karamchari

Signature of Lab technician

Red

Yellow

Black

White

Blue

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Incharge Signature…………………..

Date……………………….

 

 

 

APPENDIX 3

LOG OF BIOMEDICAL WASTE AUTOCLAVED

 

Date

Time Start

Time End

Temp & Pressure

Control tape

No.of Bags Autoclaved

Autoclaved by

Remark

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

Regents  for Estimation of  Alkaline Phosphatase:

 

1.

Carbonate Bicarbonate Buffer

=

Sodium bicarbonate (NaHCO3) = 1.68 gr

=

Sodium Carbonate (Na2CO3) = 3.15 gr

=

Dissolve in 500ml Distilled water

2.

Substrate

=

Phenylphosphate Disodium Salt Dihydrate (C6H5Na2O4P2H2O) = 1.09 gr

=

Dissolve in 500ml Distilled water

3.

0.5N NaOH

=

25 ml NaOH Solution 10N = Dissolve 500 ml distilled water

4.

Sodium bicarbonate 0.5N (NaHCO3)

=

Sodium Bicarbonate = 21 gr

 

 

=

Dissolve in 500 ml Distilled water

5.

4-Amino Antipyrine (C11H13N3O)

=

4 Amino Antipyrine = 3.0 gr

=

Dissolve 500 ml Distilled water

6.

Potassium Ferricynide K3Fe (CN)6

=

PotasiumFerricynide = 12 gr.

=

Dissolve 500 ml Distilled water

 

Preparation of STD

=

10ml Distilled water

                +

100 ml Phynol Solution

 

   

SGOT Aspartate  Aminotransferase (AST):

 

1.

Substrate

=

Aspartic Acid = 2.66 gr

=

 aKetoglutaric Acid = 30 mg

=

1.8 ml 10N NaOH

=

Makeup 100ml by stock Buffer

 

Stock Buffer = Potassium Dihydroge Phosphate = 2.7 gr

Phosphate Buffer = Disodium hydrogen = 11.3 gr

= Dissolve 1000 ml Distilled water

2.

2, 4 – Dinitrophenylhydrazine (DNPH) (C6H6N4O4)

=

DNPH = 100mg +50 ml HCl

=

Dissolve in 500 ml Distilled Water

3.

0.4 N NaOH

=

NaOH 10N = 80 ml

=

Dissolve in 2000 ml Distilled Water

= Dissolve in 1200 ml Distilled Water

 

Preparation of STD

Step (1)

=

=

Sodium pyruvate = 22 mg

10ml Phosphate Buffer

 

Step (2)

=

=

1ml stock Standard

4ml Phosphate Buffer

(Use 100 ml)

 

    

SGPT Serum glutamate – pyruvate transaminase (ALT):

 

1.

Substrate

=

DL Alanine = 1.8 gr

=

 aKetoglutaric Acid = 30 mg

=

18 ml Distilled water Dissolve

=

100 ml makeup by Phosphate Buffer

=

PH = 7.0

2.

DNPH

=

DNPH = 100mg

=

50 ml HCl

=

Dissolve in 500 ml Distilled Water

3.

0.4 N NaOH

=

NaOH 10N = 80 ml

=

Dissolve in 2000ml Distilled water

Preparation of STD:

 

Step (1)        (Stock Standard)

=

Sodium pyruvate = 22 mg

=

10 ml Phosphate Buffer

 

Step (2)        (Use 100 ml)

=

1 ml stock standard

=

4 ml phosphate Buffer

 

 

Reagents for Estimation of Serum Bilirubin:

 

Diazo A

Sulfanilic Acid

=

1.0 gr

Hydrocloric Acid HCl

=

15 ml

Dissolve in 1000 ml

=

Distilled Water

Diazo B

Sodium nitrite

=

500 mg

Dissolve in 100 ml

=

Distilled Water

Diazo Reagents

Diazo A 10 ml

=

mixed

 

  

Regents  for Estimation of  Serum Amylase:

 

1.

Buffered Starch Substrate (PH 7.0)

=

Dry anhydrous disodium hydrogen phosphate = 2.66 gr

=

Benzoic Acid = 800 mg.

 

Both reagents dissolve in 50 ml Distilled water. Bring to the boil. Mix 40 mg of soluble starch in 5-10 ml. Cold distilled water in a beaker and add it all to the boiling mixture, rinsing the beaker out with additional cold water. Continue boiling for 1 min. then cool to room temperature and dilute to 100 ml. Keep the solution at 40 and prepare freshly each month.

2.

Stock iodine Solution (0.1N)

=

Pure Sublimed iodine = 1.3 gr.

=

Potassium iodine = 2.4 gr.

=

Both reagents dissolve in 20 ml Distilled water and make to 100 ml Distilled water.

3.

Working iodine solution

=

Potassium fluoride = 5.0 gr.

 

Dissolve in 10ml Distilled water and make to 100ml with Distilled water. Store at 40 in a brown bottle.

 

 

Undergraduate Lab

  • Undergraduate lab is cleaned daily in morning by the assigned person.
  • Reagents/ chemicals for the practical class are prepared one day in advance.
  • Setting of the lab is done, all reagents and glass wares are kept at their respective places.
  • Lab is cleaned again after the practical class is over.
  • Glass wares are cleaned and kept at their respective places.

Tissue Culture Lab

Part-(A):- Personal Protective Equipment


  • Lab coat or lab gown.

  • Safety glasses if handling eye hazards.

  • Gloves if handling risk group 2 microbes or clinical samples.

  • Proper enclosed footwear 5. Hair tied back if long.


Part-(B):- Potential Hazards and Safety precautions


  • Ultra violet lights in tissue culture room pose a burn hazard to skin, and blindness hazard to eyes. UV light can cause DNA damage leading to cancer. Ensure UV lights are off before starting work.

  • Ethanol and other disinfectants can be toxic and/or flammable. Know the risks associated with each reagent before you start work.

  • Tissue culture may pose a biohazard risk depending on what it is used for, e.g. when used to propagate viruses, this can be a serious human health risk. Know the risks associated with the biohazards before starting work.

  • Tissue culture work is typically done inside a biosafety cabinet or other protective cabinet. Ensure you also read the correct risk assessment and SOP for the type of cabinet you are using e.g. consult the SMB SOP for Biosafety II cabinets.

  • The CO2 gas used in the tissue culture incubator can pose an asphyxiation or toxicity risk if inhaled. Ensure you have also read and understood the risk assessment and SOP for working with compressed gases. If you have any evidence that a CO2 leak is occurring or imminent, leave the room immediately and consult your supervisor and/or the room custodian.


Part-(C):- Procedure


  • Check first with the room custodian before starting work. The custodian will instruct you on which hood and incubator to use – follow these instructions, and do not use other hoods/incubator.

  • Ensure that the door to the tissue culture room is kept shut at all times. When in the tissue culture room, wear disposable gloves and a gown with long sleeves and cuffs. The gown is to be kept inside the tissue culture room and MUST NOT be used for general lab work. Do not wear your regular lab coat/gown in the tissue culture room – this can lead to contamination problems.

  • Wipe down work area and equipment with 80% ethanol before starting.

  • Everything used in the tissue culture lab must be labelled, including your initials, the date, and the cell line. For cell passages, it is usually best to include passage # and split ratio for your own information.

  • If you have a lot of tubes, etc. that are not for long-term use, you can avoid labelling them all, but ONLY IF you dispose of properly immediately after you are done. At no time can you leave unlabeled materials in the lab.

  • Turn the UV light on and fan on for 20 minutes before using the hood.

  • Turn UV off before starting work, but leave fan on.

  • UV irradiate for 20 minutes between cell types and strains and when finished.

  • Everything going into hood must be swabbed with EtOH including gloved hands.

  • Dry media bottles thoroughly if they have been taken out of the water bath (this water is a great source of contamination). Swab with EtOH, especially at the neck and bottom before placing in hood.

  • Clean spills of culture or media immediately with EtOH.

  • Ideally, hoods should be cleaned thoroughly approximately every 6 weeks, depending on usage. Swab interior with 70% ethanol. Wash the interior of the cabinet with disinfectant/detergent, including inside of glass panels, roof and walls. Swab interior again with 70% ethanol. Replace front panels and UV irradiate for a further 20 minutes.

  • Regularly check the temperature and CO2 levels displayed on the incubator and also the CO2 levels of the cylinder, notify room custodian if temperatures are incorrect or CO2 levels are low.

  • Swab gloved hands with EtOH before opening the incubator.

  • Dispose of unwanted flasks appropriately ASAP, to minimize clutter and contamination risk (see Risk assessment & SOP ‘Disposal of biological wastes’).

  • Clean spills of culture or media immediately with 70% ethanol– use the appropriate disinfectant for the biological agents present (e.g. ethanol is insufficient to kill viruses).

  • All cell lines should be inspected regularly for microbial contamination.

  • Check the water level (top up with autoclaved water only) and check for any contamination in the water bath for example turbidity or fungal growth.

  • Water should be changed monthly (more often if heavy usage). Spray the inside of the bath with EtOH before refilling with autoclaved water.



Part-(D):- – Disposal


  • Cell culture liquids must be disposed of by autoclaving, or treated freshly prepared 1% bleach. Anything that has been exposed to cell culture should also be similarly treated. After decontamination by these methods, liquids can then be disposed of via the sink.

  • Do not leave waste media in the tissue culture room and do not over fill the biohazard bag. When the bag is 2/3 full, tie and take to be autoclaved on Level 2 and please replace the bag immediately!

  • Any spills must be cleaned up immediately. Depending on the nature of the spill, clean up as described in SOPs for Biohazard Spills, Flammables, Corrosives, or Toxic Substances.

  • Any large spills of hazardous materials (>1L) or incidents resulting in injury must be reported to your supervisor immediately and via the online incident report form within 24 h. Near misses (dangerous situations not leading to an incident) should also be reported.


General Laboratory Safety Procedures

DOs

·         Know the potential hazards of the materials used in the laboratory. Review the Safety Data Sheet (SDS) and container label prior to using a chemical.

 

·         Know the location of safety equipment such as telephones, emergency call numbers, emergency showers, eyewashes, fire extinguishers, fire alarms, first aid kits, and spill kitswhichcanbefoundonallcampuses(IUPUIdoesnotprovidelaboratoryspillkits).

 

·         Review your laboratory’s emergency procedures with your Principal Investigator or Lab Supervisor to ensure that necessary supplies and equipment are available for responding to laboratory accidents.

 

·         Practice good housekeeping to minimize unsafe work conditions such as obstructed exits and safety equipment, cluttered benches and hoods, and accumulated chemical waste.

 

·         Wear the appropriate personal protective apparel for the chemicals you are working with. This includes eye protection, lab coat, gloves, and appropriate foot protection (no sandals or open toed shoes). Gloves must be made of a material known to be resistant to permeation by the chemical in use.

 

·         Shoes must cover the entire foot. Open toed shoes and sandals are inappropriate footwear in laboratories. Fabric and athletic shoes offer little or no protection from chemical spills. Leather shoes with slip-resistant soles are recommended.

 

·         Street clothing is to be chosen so as to minimize exposed skin below the neck. Long pants and shirts with sleeves are examples of appropriate clothing. Avoid rolled up sleeves. Shorts (including cargo shorts), capris and, miniskirts are inappropriate clothing in laboratories. Tank tops, sleeveless shirts and midriff-length shirts are not appropriate if not covered by a full-length laboratory coat and must not be worn if wearing an apron alone. Synthetic fabrics must be avoided in high-hazard areas where flammable liquids and reactive chemicals are utilized.

 

·         Contact lenses are not recommended but are permitted. Appropriate safety eyewear is still required for those that use contact lenses. Inform the lab supervisor of the use of contact lenses.

 

·         Wash skin promptly if contacted by any chemical, regardless of corrosively or toxicity.

 

·         Label all new chemical containers with the “date received” and “date opened.”

 

·         Labelandstorechemicalsproperly.Allchemicalcontainersmustbelabeledtoidentify the container contents (no abbreviations or formulas) and should identify hazard information.Chemicalsmustbestoredbyhazardgroupsandchemicalcompatibilities.

 

·         +Use break-resistant bottle carriers when transporting chemicals in glass containers that are greater than 500 milliliters. Use lab carts for multiple containers. Do not use unstable carts.

 

·         Use fume hoods when processes or experiments may result in the release of toxic or flammable vapors, fumes, or dusts.

 

·         Restrain and confine long hair and loose clothing. Pony tails and scarves used to control hair must not present a loose tail that could catch fire or get caught in moving parts of machinery.

 

DON’Ts

·         Eat, drink, chew gum, or apply cosmetics in rooms or laboratories where chemicals are used or stored.

 

·         Store food in laboratory refrigerators, ice chests, cold rooms, or ovens.

 

·         Drink water from laboratory water sources.

 

·         Use laboratory glassware to prepare or consume food.

 

·         Smell chemicals, taste chemicals, or pipette by mouth.

 

·         WorkaloneinthelaboratorywithoutpriorapprovalfromthePrincipalInvestigator or Lab Supervisor. Avoid chemical work or hazardous activities at night or during off-hours. Have a partner for assistance (use the “buddy-system”) at night or during off-hours.

 

·         Leave potentially hazardous experiments or operations unattended without prior approval from the Principal Investigator or Lab Supervisor. In such instances, the lights in  the laboratory should be left on and emergency phone numbers posted at the laboratory entrance.

Safety Procedures In Research Laboratory

  • A science laboratory is a place where basic experimental skills are learnt only by performing a set of prescribed experiments. Safety procedure usually involves chemical hygiene plan and waste disposal procedures and significant physical and health hazards associated with the specific type of research and instruction in specific procedures that researchers should use in order to prevent and limit exposure to the health hazards in that workplace. Safety is a learned behavior that must incorporate into our instructional plans.


    v  PERSONAL PROTECTIVE EQUIPMENT AND CLOTHING

    Personal protective equipment is used in order to protect ourselves when working with chemical hazards. Common examples of personal protective equipment include: lab coats, footwear, gloves, safety goggles and glasses, face shields, hard hats and respirators.


    ·         Lab Coat

    The primary purpose of a lab coat is to protect against splashes and spills. A lab coat should be nonflammable and should be easily removed. Lab coats should be buttoned when in use. Rubber coated aprons can be worn to protect against chemical splashes and may be worn over a lab coat for additional protection.


    ·         Footwear

    Leather shoes which completely cover the toes, heel and top of foot provide the best general protection. The shoes must be made of water proof materials. The shoe must have a nonslip sole firmly attached to the foot. Sandals, sneakers, perforated shoes, open-toed shoes etc. do not provide adequate protection in case of spills.


    ·         Gloves

    When handling chemical, physical, or biological hazards that can enter the body through the skin, it is important to wear the proper protective gloves.


    ·         Eyewear

    Safety goggles provide the best protection against chemical splashes, vapors, dusts, and mists. Contact lenses should not be worn during any investigations using chemicals (even if you are wearing goggles).


    ·         Face Shields

    A face shield should be worn whenever there the entire face needs protection. Face shields can protect against impact, dust, particulates, and splashes to the face, eyes, and throat.


    v  EMERGENCY EQUIPMENT

    Research laboratory is equipped with a wide range of emergency equipment that can be invaluable in case of an accidental exposure to or a fire or explosion involving a hazardous reagent.


    ·         Fire Extinguisher

    Periodically check the date on the fire extinguisher to make sure that the extinguisher is full and the extinguisher is in good working order.


    ·         Eye Wash Stations

    Eye wash stations consist of a mirror and a set of bottles containing saline solution that can be used to flood the injured eye with water. The eye wash station is intended to allow us to flood the eye with a continuous stream of water.


    ·         Safety Showers

    Emergency shower are intended to provide on-the-spot cleansing when a chemical or solvent has been spilled.


    ·         First- Aid Kit

    A first aid must be readily available in science laboratory for use during accidents and emergencies. It is equipped with tincture of iodine, ferric chloride (alcoholic), burnol, savlon, cotton, sodium bicarbonate solution, adhesive plaster, bandages and scissors. Periodically inspect and restock the first aid kit.


    ·         Fume hood

    A fume hood is a type of local ventilation device that is designed to limit exposure to hazardous or noxious fumes, vapors or dusts.


    ·         Exhaust fans

    Exhaust fans must be fitted near the ceiling of laboratory for speedy removal of waste gases to keep the laboratory free from polluted air.


    v  HOUSEKEEPING

    The area is kept clean and items are stored in appropriate areas to ensure the safety of students. If everything is organized then labs will be more productive and cost effective.


    ·         Work area

    Work areas should be kept clean and free from unnecessary chemicals and obstructions. Reagents and chemicals to be used frequently are kept on reagent shelves. Reagent bottles are arranged in a definite order on these selves. All sensitive electronic equipment should be placed safely on table or within bag under table so that expensive damage can be avoided. We should not block access to emergency equipment (i.e. fire extinguishers, eyewashes, etc.), emergency shut-offs, and utility controls (i.e. electrical panels). When the fire alarm sounds we must evacuate the building via the nearest exit. Extinguish all flames and turn off all equipment before leaving.


    ·         Labeling samples and materials

    All containers containing chemicals or solutions of any kind that are retained between laboratory sessions must be labeled with full chemical name and hazard classification. The label must also contain the date and the name of the responsible person. Each laboratory must maintain chemical inventory that should be updated at least once in a year.


    ·         Chemical Handling and storage

    When dispensing chemicals, we should read the label carefully before starting the experiment. To avoid contamination and possible violent reaction we should never return unwanted chemicals to their container. We should not put dropper into a supply bottle. To avoid overheating and spurting never add water to concentric acid instead always add acid to water slowly with stirring. No pipetting should be done by mouth; we should use a pipette bulb or other pipetting device. Pipette must be used in a vertical position. Always dispense and dilute concentrated acid into a fume hood. Spilled chemicals should be cleaned up immediately and disposed of properly. Acids and  corrosive chemicals should be neutralized with soda ash (sodium carbonate) or sodium bicarbonate and spillage of alkali be neutralized by covering with dry sand. Chemicals should be stored in their original containers. We should not store chemical containers on the floor.


    ·         Hot equipment and glassware handling

    All glassware should be inspected before use and any broken, cracked, or chipped glassware should be disposed of in an appropriate container. All hot equipment should be allowed to cool before storing it. All glassware must be handled carefully and stored in its appropriate place after use. All glass chemical containers should be transported in rubber or polyethylene bottle carriers. Only borosilicate (Pyrax, Kimax etc) containers should be used for heating solutions. We should never heat a closed system such as a sealed test-tube or closed bottle. When a burner or hot plates are used we should always wear goggles and an apron to protect our eyes and clothing. We should never leave a hot plate unattended while it is turned on. Handle hot equipment with safety gloves and other appropriate aids but never with bare hands. We should keep our head, hands, hair, and clothing away from the flame or heating area, and turn heating devices off when they are not in use. Gas burners should be lit only with a spark lighter. Make sure all heating devices and gas valves are turned off before leaving the laboratory.


    v  UNAUTHORIZED ACTIVITY

    ·         Horse Play

    Horseplay in the lab is very dangerous. Unauthorized person should not allow entering in the laboratory.


    ·         Food, drink and Smoking

    No food or drinks of any kind are allowed in the laboratory. Any food or drink brought to the lab must remain in the carrying bag until they leave. We should not store food and drinks in laboratory refrigerators. Wash hands frequently throughout the day and before leaving the lab to avoid carrying toxic materials. Smoking is banned throughout the college and it is never allowed in any laboratories. In addition, do not apply cosmetics the laboratory. Never smell or taste chemicals or touch them with bare hands.


    v  WASTE DISPOSAL

    Use water taps only when required and should not waste any water. It is essential to clean the sink regularly. We should never put broken glass or ceramics in a regular waste container. Use a dustpan, a brush, and heavy gloves to carefully pick up broken pieces, and dispose of them in a container specifically provided for this purpose. Hazardous chemical waste including solvents, acids, and reagents should never be disposed of down sewer drains. Waste must be separated and disposed of in the proper waste containers.

Operation and Calibration of pH Meter

  • Objective:

To lay down the procedure to perform the Operation and calibration of pH meter

  • Key functions of pH meter

On/Off press the On/Off button of the pH meter to switch on the meter and switch off the meter.

CAL/MEAS:

HOLD/ENTER:

MI (memory input): Press MI to capture the measured reading of the pH with its corresponding temperature values and store them in memory.

Scroll through each SETUP and its sub group menu.

MR (memory recall): Press MR to retrieve the stored data from memory.

SETUP: Press SETUP  to activate the parameter setting menu to customize meter configuration, view calibration points and electrode offset data, select auto power off, reset meter and clear memory.

MODE: Select the measurement parameter option between pH with temperature.

  • General

Electrode Cleaning: Electrodes, which are mechanically intact, can often be restored to normal performance by one or combination of the following procedures.

Salt Deposits: Dissolve the deposit by immersing the electrode in raw water for ten to fifteen minutes. Then thoroughly rinse with de-ionized water.

Oil / Grease Films: Wash electrode pH bulb in a little detergent and water. Rinse electrode tip with de-ionized water.

Clogged Reference Junction: HeatHhhhhhhhhhHeat a dilute KCI solution to 60-80 C. Place the sensing portion of the pH electrode into the heated KCO solution for approximately 10 minutes. Allow the electrode to attain room temperature while immersed in some unheated KCI solution.

Electrode Activation: In case of dehydration/electrode showing sluggish response, the bulb can be re-hydrated by immersing the electrode in an ideal storage solution (e.g. buffer pH 5.00/5.01 solution) for 1-2 hours. If this fails, proceed for the electrode rejuvenation.

  • Operation of pH meter to determine pH of solution

Press ON to switch on pH meter. The MEAS annunciator appears on the top center of the LCD. The ATC indicate appears in the lower right hand corner to indicator automatic temperature compensation.

Rinse the electrode with de-ionized or distilled water before use to remove any impurities adhering to the probe body.

Maintain the temperature of the sample to 25C + 2C, unless otherwise specified in the individual monograph and dip the electrode along with the temperature sensor into the sample.

When dipping the electrode into the sample the sensor or the glass bulb of the electrode must be completely immersed into the sample, stir the probe gently in the sample to create a homogeneous sample.

Allow time for the reading to stabilize.

If the READY indicator has been activated the READY annunciator lights when the reading is stable

Press the HOLD to freezing of the measured reading and Press the ENTER to confirm the measured reading.

Record the observed values of pH and Temperature in the respective test data sheet or technical information sheet.

Wash the electrode in de-lonized water after use and store the electrode in storage solution or pH buffer 5.00/5.01 or 7.00/7.01 buffer solution.

  • Calibration of pH meter

Before starting the calibration make sure that the correct measurement mode is selected.

Wash the electrode thoroughly with de-lonized water or a rinse solution. Do not wipe the electrode, this causes a build-up of electrostatic charge on the glass suface.

Maintain the temperature of the buffers to 25C + 2C, unless otherwise specified in the individual monograph and dip the electrode along with the temperature sensor into the buffers. 

Perform the five point calibration buffer. The end of the electrode must be completely immersed into the sample. Stir the electrode gently to create a homogeneous sample.

Press CAL/MEAS key to enter pH calibration mode. The CAL indicator will be shown. The primary display will show the measured reading while the smaller secondary display will indicate the pH standard buffer solution reading.

Wait for the measured pH value to stabilize.

Press HOLD/ENTER key to confirm calibration. The meter is now calibrated to the current buffer.

Fist rinse the electrode with de-ionized water, which is followed by next buffer solution and place it in the buffer solution.

Follow steps 5.5.5 to 5.5.9 for additional calibration points.

When all the calibration points set in the unit configuration set up are completed the meter returns to the measurement mode automatically. However calibration can be terminated without completing the number of points as set in the unit configuration. It can be done by pressing CAL/MEAS to return to pH measurement mode. Record the calibration details and Temperature.

Change the calibration buffer every week or whenever required and record.

  • Determination of pH Electrode Slope

Press the SETP key to enter Set Up mode.

Press the MI/▲ or MR/▼ key to scroll through subgroups until you view parameter P3.0.

Press the HOLD/ENTER key. The display show the electrode offset value. If you have not calibrated at any buffer, the primary display show ‘-----‘

Press the HOLD/ENTER key to proceed to electrode slope display.

The display shows electrode slope in percentage. Slope displayed will be the current slope of the slope zone to which a measurement is made or calibration is done. Record the electrode slope.

  • Storage

Always keep the electrode bulb wet, preferably in the pH 5.00/5.01 buffer. Raw water is also acceptable storage media, but avoid storage in de-ionized water.

During handling of pH meter, check if electrode is broken, replace the same with new electrode and enter the details in instrument usage log book for pH meter.

  • Electrode Rejuvenation

If the electrode fail to give appropriate result during calibration, the same shall be Rejuvenated as mentioned below:-

Dip and stir the electrode in ethyl alcohol for 5 minutes.

Leave the electrode in tap water for 15 minutes.

Dip and stir the electrode in concentrated acid (e.g. Hydrochloric Acid, Sulphuric Acid) for 5 minutes

Leave the electrode in tap water for 15 minutes.

Dip and stir the electrode in strong base (Sodium Hydroxide) for 5 minutes.

Leave for 15 minutes in raw water.

Test with standard calibration buffer solution.

If the electrode still fails to restore the normal electrode response, the electrode rejuvenation steps (5.7.1.3 – 5.7.1.6) shall be repeated.

If the response does not improve, then the electrode has completed its useful life and same shall be replace with a new electrode.

Record the Electrode Rejuvenation details.

Electronic Balance

  • All balances should be located on a sturdy bench, free of vibration and drafts. The balance should be leveled. Adjust the position of the bubble in the level indicator by using the screw feet at the bottom of the instrument. The bubble should be in the center of the circle. Leave balance on at all times to maintain thermal equilibrium.

  • Clean balance after each use using a non-abrasive cleaner or a lint-free brush. Use only clean weighing vessels for weighing. Disposable, plastic weigh boats are most convenient for weighing of various chemicals.

  • Place weighing vessel (use smallest possible container) squarely in the center of the weighing pan.

  • Tare the instrument by pressing the re-zero or tare position on the control bar of the instrument. Instrument should read zero before adding substance. The weight of the container is now tared out.

  • With a clean scoop, add a small portion of sample at a time to avoid overshooting target weight. Avoid touching weigh vessel or pan. After reaching target weight, remove weigh vessel with sample and re-tare (re-zero) balance by pressing the tare (re-zero) position on the control bar.


Microscope

Procedure:

1.      Ensure that the microscope and its surrounding area is clean.

2.      Plug the microscope power cord in to electrical out let.

3.      Turn on the microscope by switching it on from main switch and two other switches one at back of microscope and at left lower side of microscope. This will switch on the light of microscope also.

4.    Set the intensity of light to the lowest setting using illumination control knob present at bottom in front side of microscope.

5.      Place the specimen slide on the stage.

6.      Rotate the nosepiece to move the objective (10X/20X/40X/60X) into working position.

7.   Change the position of nosepiece by using the knob present on left lower side to focus on the specimen. See through the eyepiece to do so

8.    Next, focus the specimen through the left eyepiece by turning the eye tube. Cover the right eyepiece while doing this and be sure to focus with the left eye tube only, without using the focusing knob.

9.      Observe the slide.


Cleaning and Maintenance

1.      Whenever lack of contrast, cloudiness or poor definition is encountered, Clean the lower magnification objectives and optical surfaces with a lint free cloth or lens tissue moistened (not wet) with ethanol.

2.      Avoid excessive use of solvent for cleaning.

3.      Cover the microscope always with dust cover, whenever the microscope is not in use.

4.      Wipe the bottom of Oil immersion lens of a fast absorbing tissue paper before and after using the lens.


Zeiss Optical Fluorescence Microscope

  • Materials allowed on the Microscope:

1.      No restriction on samples unless they are non-standard biological samples requiring level 3 or greater isolation rooms.

2.      For biological samples, prior approval from respective supervisor is required.

3.      Oil immersion microscopy can be carried out.

4.      It is better for the candidate to know the excitation frequency for the sample but it is not a mandate.


  • Authorization Procedure:

Authorization will be given by the chairperson. The candidate must have the understanding of the general components of the Microscope; i.e. the fluorescence source, the different filters, the magnification tool and other basics. An understanding of the data capturing software. Data storage and transfer is permitted using a CD/DVD. No USB drives allowed. Hands on experience using the standard sample is carried out a couple of times. Safe switching on' and 'switching off' of microscope.


  • Standard Operating Protocol:

The standard rule for the microscope's use is to complete one's work and leave the microscope back to its initial state; made ready for the next operator's use.

1.      When the Microscope is off, all the electrical switches must also be off. The eyepiece cover, the plastic encapsulation must be in the right place. The lever that helps switch from the camera to the eyepiece must at all times in a switched off mode be directed to the eyepiece.

2.      When using the microscope for normal fluorescence microscopy, the mains must be switched on first. The computer must not be switched on along with the microscope. First, the microscope's power module must be switched on, then the microscope and then the Fluorescence power source (Xcite). Fluoroarc is another module that sits just below the Microscope's power source. This is the secondary Fluorescence source. As long as the 'Xcite' source is functional, 'Fluoroarc' must always be kept off.

3.      Before turning ON the microscope, always check the log book for last user entry. If the fluorescence lamp was used in the past hour, allow it to cool. Also, report any abnormalities in the log book to the system owner.

4.      Before placing the sample on to the microscope table and before switching the system off, please ensure that the magnification has not been set to max or minimum; i.e. 100X or 1X.

5.      While switching off, the system must be left as found by the user before switching it on.


  • Negligence:

1.      If the last user for instance forgets to write proper details of his/her run in the log book

2.      If after use, the eye-piece cover or the microscope encapsulation has not been put on properly

3.      If the table-top has not been cleaned properly after the experiment.

4.      If the user does not operate the hardware or the software properly and is visible in the form of remnants after the experiment or not saving data appropriately thus putting other users' data at risk

5.      If the fluorescence lamp is left on. These components even though long lived, are very crucial to the operation of other experiments as well. Leaving it on results in unnecessary heating of the lamp not allowing the next user to use it.

6.      If the user does not take care of the lenses and does not report a mishap. This negligence might result in others loosing precious working hours.


This negligence put the system at absolute risk and shows direct negligence towards other users.


  • Manual

An electronic copy of the maul as provided by the manufacturer is available on the computer supporting the Microscope's data capture.


  • Logbook

The Logbook clocks in every activity by the authorized users.


  • First Level Tool Maintenance

The first level maintenance involves a weekly check of the vital part of the microscope namely,


  1. Eye piece, Lens - for dirt
    1. If dirt is found, the chairperson must be notified who will then schedule an appointment with the Zeiss engineer for a cleanup.
    2. The Zeiss instructed not to try and clean up any of the internal components because each one is extremely expensive.

  1. Fluorescence lamp
If the intensity of the lamp or any of its filters is seen to be different, it must be reported.

Cooling Centrifuge

Procedure:

Identify the speed and duration at which you wish to centrifuge samples PRIOR to using this instrument.


1.          Check the rotor you intend to use and be certain that the rotor is rated for the speed at which you would like to use it.

2.         If the rotor is not capable of being operated at the target speed, you will need to identify the rotor that is capable of being used at the desired speed, and then transfer your samples to a centrifuge tube that will fit and rebalance the samples, remembering to include the lids when balancing.

3.        Place the rotor in the centrifuge Check the name of the rotor and confirm the target speed.  Locate the correct lid for the selected rotor and place it beside the centrifuge.

4.     Place the samples into the rotor.  If the samples do not all have the same mass, place samples with the same mass on opposite sides of the rotor.

5.       Once the samples are loaded, check the rotor type again, confirm that you have the correct lid for the selected rotor and then screw the rotor lid onto the spindle.  The rotor lid set screw turns opposite to “normal” screws.

6.        Once the rotor lid is secured, close the centrifuge lid and set the desired temperature, the desired speed and the desired spin time.

7.        When everything is set, press the “Start” button and wait for the instrument to ramp up to the desired speed.

8.       Any large vibration in the instrument or irregular noise may indicate that something has gone wrong with the run.  Press the “Stop” button and move away from the instrument. Immediately contact a faculty member who can assist you BEFORE you open the lid to the centrifuge.

9.            If no problems are detected as the instrument reaches the desired speed, you may leave the area until the run is complete.

10.   When the run has completed and the rotor has come to a complete stop, you may unscrew the rotor lid and carefully remove your samples.

11.    Should a sample vial have leaked during the run, you MUST IMMEDIATELY clean the spill and dry the rotor. It is easy to miss spilled liquids in the bottom of the positions of the rotor, however, this liquid will cause an imbalance in the subsequent run that may lead to catastrophic failure.

12.     Remove the rotor from the centrifuge and place in the appropriate box adjacent to the unit.

13.     Should a sample vial have leaked during the run, you MUST IMMEDIATELY clean the spill and dry the rotor.

14.     It is easy to miss spilled liquids in the bottom of the positions of the rotor, however, this liquid will cause an imbalance in the subsequent run that may lead to catastrophic failure.

15.    Clean the area around the centrifuge with 70% ethanol (denatured) and wipe dry. This MUST be done following EVERY run.  Failure to clean up after yourself may ruin subsequent users’ experiments and/or facilitate the spread of contamination throughout the department.


Safety: Wear gloves throughout the procedure.  Do not leave the instrument.


Troubleshooting: Inform your supervisor or person responsible for that area of any issues


Operating and Cleaning of Distilled Water Unit

  • Objective

To lay down the procedure for Operating and cleaning of distilled water unit


  • Procedure

Connect the water inlet pipe to the tap.

Switch on the main supply

Keep an empty clean water container below the water outlet pipe to collect the distilled water.

Switch on the unit.

Collect about 500 ml of distilled water initially in the tank and discard it. Then collect the required amount of distilled water by operating the unit

Analyse the distilled water every week and report.


  • Cleaning of the unit

Dismantle the unit with the help of maintenance person

Clean thoroughly the storage container.

Put about 500 ml of dilute (5%) Hydrochloric Acid in the distillation unit.

Keep the Hydrochloric Acid in the unit for about 12 hours.

Wash away the acid with tap water very thoroughly.

Start the unit and collect the water to about 2 Lit. discard the collected water

Clean the unit every month.


Milli-Q Water System from Millipore

  • Objective

    To describe a procedure for operation of Milli-Q water system from Millipore
  • Procedure

Operating Procedure

Ensure the area is clear.

Ensure that water supply is continuing

Pressure gauge shows the water circulation.

Pressure should be more than 1.5 kg /cm2.

Instrument has three parts:

Put on the main switch connected to the system.

Light glows to indicated the power supply to the instrument.

Press operates / stands by mode, displays “pre operate” in Elix.

Water treated in Elix System collect automatically in “reservoir”.

Water again to filter through Milli-Q system.

Press operates / stands by mode, displays “pre operate” in Milli-Q

Collect Milli-Q water through the outlet.

Press the key stand by display shows “Standby”.

Never put off the power supply to the instrument.

Fixed Wavelength Fluorometer

The Quantech Life Sciences Fluorometer has the ability to measure fluorescence at two emission wavelengths (340 and 450nm) during excitation by UV light at 280 nm.

The unit should be equipped with 2 sets of filters (one for use; one spare). There should be one narrow band excitation wavelength filter at 280nm (280NB), one narrow band emission filter at 340nm (340NB), and one narrow band emission filter at 450nm (450NB) in each set; there should also be one blocking filter.


Filter Placement

The positioning of the filters are as follows:

  • When facing the instrument for operation with the cover open, the 280nm excitation filter fits into the filter slot at the 6 o’clock position.

  • The emission filters (either 340nm or 450nm) fit into the filter slot at the 9 o’clock position.

  • The blocking filter fits into the filter slot at the 12 o’clock position; it and the 280nm filters should remain in place (not be moved).


Startup

  • At least 1 hour before the first sample is to be analyzed, the instrument is turned on (switch at the back of the unit near the power cord). The instrument will go through a start up sequence and display a 15 minute countdown.

  • After 15 minutes has elapsed, the display will show Menu .
    Push the

<Right Arrow> once until the display reads Advanced Functions . Push the <Enter> key.  <Date and Time> will appear.  Press the <Right Arrow> key until <UV Lamp Options> is displayed. Press <Enter>. The display will read <UV Lamp Installed?> with <No> underneath.


<Up/Yes> key to turn the UV lamp on and press <Enter>. You now must wait 20 minutes for the UV bulb to warm up. Menu should once again be displayed.

  • After at least 20 minutes, press the <Right Arrow> to select Advanced functions (Press <Enter>).  Press the <Right Arrow> until <Manually Set Gain and PMT Voltages>. Press <Enter>. The display will show <Turn off Auto Gain?> Press the <UP/Yes Arrow>and press <Enter>. Press the<Up/Yes Arrow> until Gain= 100 is displayed and press <Enter>. .


  • Menu will be displayed. Press the <Right Arrow> to select

Advanced Functions (Press <Enter>) and <Right Arrow>until <View Diagnostic Information> is on the display.  When Raw PMT….

Hardware id displayed, press the <Left Arrow> key to select <Raw fluorescence>, and press the <Right Arrow> to select <UV Lamp>. Press the <UP/Yes> key to select the gain at 100. The raw fluorescence value should start fluctuating.


Drawing Samples for Analysis

Note: SAMPLES FOR THE FLUORESCENCE INTENSITY RATIO SHOULD ONLY BE TAKEN FROM BOTTLES FIRED IN AREAS OF ELEVATED FLUORESCENCE IN THE CTD TRACE.


  • A 20 ml sample is acidified with 20L of 10% HCl in a 20 mL scintillation vial equipped with a foil or Teflon lined cap and stored at 4oC.

  • Samples are drawn from the same cups as used for the LISST with the following exception – pour the contents of sample cup #2 into sample cup #1 (after the LISST sample from cup #2 has been successfully analyzed). This creates an integrated sample from the one niskin bottle/sample depth.

  • Draw duplicates (labeled “sampleID”-1 and “sampleID”-2) from the integrated cup. Acidify the sample. (NB – It is easier to add the acid to the vial first and then the sample to ensure adequate mixing).

  • Place approximately 3.5 mL of sample into a methacrylate disposable cuvette and allow the samples to approach room temperature; a cold sample will effect the fluorescence measurement. Refrigerate the remaining sample for storage, or discard if not to be archived.

                                                  

Sample Analysis

  • Place the cuvette into the holder of the Quantech fluorometer. Make sure the 340nm emission filter is in the proper filter holder position (9 o’clock). Close the lid.

  • Wait a moment (count to five) and glance at the display. As the reading is not stable, glance at the reading and record the first number you see in the back of the fluorometry notebook under a column labelled “340”. Repeat this procedure twice more for a total of three (3) times (for example, sample ID BM550107-2 had 340 nm readings of 55, 63, 44).

  • Open the cover of the fluorometer and replace the 340nm filter with the 450nm filter. Close the cover, count to five, and read the display three times in the same way as for the 340nm wavelength (for example: sample ID BM550107-2 had 450 nm readings 27, 23, 33).


  • Average the three 340nm readings (eg, 55+63+44=162 / 3= 54) and the three 450 readings (eg, 27+23+33=83 / 3= 27.7)

  • Obtain the fluorescence intensity ratio (FIR) by dividing the 340 nm by the 450 nm average (eg, 54 / 27.7 = 1.95)

  • Repeat this procedure for the next samples.


Reporting Data

The FIR data are to be included with the LISST particle data in the daily report. FIR should be recorded as a number on the LISST particle graph above the corresponding LISST sample in the histogram.

Ultraviolet–Visible (UV-Vis) Spectroscopy (Hitachi)

Description of Process

The ultraviolet–visible spectroscopy (UV-Vis) utilizes light to determine the absorbance or transmission of a chemical species in either solid or aqueous state.


Personal Protective Equipment

EYE PROTECTION: Safety glasses

PROTECTIVE CLOTHING: Laboratory coat and nitrile gloves


Sample Preparation Procedure

If the sample is in the solid state, the sample should be coated on to a desired, transparent substrate. A blank sample of the transparent substrate should be brought to do the experiments.


If the sample is in solution, it should be premixed to the desired concentration and a blank sample of the solvent should be brought to do the experiment. Note that if quartz solution cells are used, they should be cleaned before and after use with the solvent that is to be used during the experiment.


UV-Vis Procedure


1.   Check inside the UV-Vis chamber to assure that the appropriate sample holder (i.e., the liquid or solid sample holder) is in place. If it is not switch it out, the correct sample holder will be in the cabinet above the equipment.

2.   Turn the UV-Vis on by pressing the “Power” button in the rear left side of the unit. The unit is not operational until a light on the front goes to a solid green color.

3.   Log onto the computer connected to the UV-Vis.

4.   Open “UV Solutions 4.2” from the desktop.

5.   Click on “Method” on the right to set the experimental parameters.


a.   Instrument Tab

i.    Change the Wavelength range settings, if applicable.

ii.   Set Scan controls to an appropriate speed. The “Slow” setting is a good choice if data is for publication.


b.   Report Tab

i.    Select parameters according to the instrument settings. Change reportformat to excel mode.


c.   Click OK.


6.   Click on the Baseline Button on the right-hand side of the screen. A pop up will prompt you to place the blank substrate in the holder. Once this is in place click OK.

7.   Once the Baseline procedure is done, take out the blank sample and put in your sample.

8.   Go to Measure at the right of the screen and click on it. The scan will begin at this time.

9.   Once the scan is finished, a pop up will come up that will allow you to scan another sample of finish. If you are running multiple samples change your sample, change the sample name on the pop-up box, and click OK otherwise click Finish.

10. Save your data to the correct file folder.

11. Turn the UV-Vis OFF by pressing the “Power” button.

12. Logoff of the computer.

13. Enter your user information into the logbook.


Data Transfer Procedure

To access your data, you can do it with directly from the program or open User Data from the desktop by selecting the appropriate file. Once your data is open go to File, Save As, and save it as .csv file. This will allow you to open the data in Excel.


Shimadzu UV-Vis Spectrophotometer (UV-1700)

Purpose:

1.1.        Operation of the Shimadzu 1700 UV-Visible Spectrophotometer.


2.           Scope:

2.1.        This machine is used to measure how much light of a given wavelength is absorbed by a liquid sample.


3.           Responsibilities:

3.1.        It is the responsibility of the lab in-charge to ensure that this SOP is performed as described and to update the procedure when necessary.


3.2.        It is the responsibility of the students to follow the SOP as described and to inform the in-charge about any deviations or problems that may occur while performing the procedure.


4.           References:

4.1.        Shimadzu UV-Visible Spectrophotometer manufacturer’s instructions.


5.           Definitions:

5.1.        Cuvette: A small, transparent vessel. This is what a sample is put into for testing in the UV-Visible Spectrophotometer.


6.           Precautions:

6.1.        N/A


7.           Materials:

7.1.        sample to be tested

7.2.        cuvettes (visible or ultraviolet)

7.3.        lab tissue

7.4.        blank solution.

7.5.        2mL pipettes


8.           Procedure:

8.1.        Open panel door and make sure cuvette holders are empty, then close the panel door.

8.2.        Turn spectrophotometer “ON” by flipping the switch on the side of the machine.

8.3.        The machine will automatically initialize and make a base line correction.

8.4.        Select method according to the requirement (wavelength scan, photometry, etc)

8.5.        Fill 2 of the same cuvettes each with about 2mL of blank solution. Hold the cuvette from the top to prevent tampering with the measurements, and wipe the sides with a lab tissue.

8.6.        Open panel door and place the cuvettes with blank solution in the cuvette holders. Make sure to use the appropriate orientation for the cuvettes you’re using. Also make sure that the cuvettes used for the autozeroing are the same cuvette you use for the sample reading. If using a standard cuvette, any orientation of the cuvette in the holder is acceptable, just make sure you wipe the cuvette’s sides. If using a micro cuvetter, the microcuvette MUST be oriented in the holder so the the 1cm path length goes from left to right.

8.7.        Press the AUTO ZERO key, then press ENTER.

8.8.        When the the Auto Zero is complete, open the panel door and remove the front cuvette.

8.9.        Do not replace cuvette in rear holder.

8.10.      Using the same cuvette style, fill an empty cuvette with about 2-ml of the sample.

8.11.      Clean the cuvette with a lab tissue.

8.12.       Place in front cuvette holder, using the appropriate orientation and close the panel door.

8.13.       Press START to take a reading.

8.14.       Record the results or press COPY for a hard copy printout.

Note: If the initial sample OD reading is greater than 1.0, the sample should be diluted until it reads below 1.0 and then multiply by the dilution factor to obtain the absorbance value.

8.15.       Open panel door and remove test sample from front cuvette holder.

8.16.       To test additional samples: Place cuvettes in front holder and press start for a reading.

8.17.       Record results, or press COPY for a hard copy printout.

8.18.       Press RETURN to bring you back. Note: This will erase your old data.

8.19.       Press FILE to return to the original screen.

8.20.       Remove cuvettes remaining in holders.

8.21.       Flip power switch located on the side, to turn off the machine.


9.           Log Book:

9.1.      Record in the log book

Shimadzu UV-VIS Spectrophotometer (UV-1800)

PPE required

Gloves, lab glasses, lab coat


Procedure

  1. Turn on UV-Vis with switch on side

  2. Allow the instrument to initialize

  3. Wait 30 min to 1 hr for the instrument to warm-up

  4. Login to the UV-Vis by pressing enter on the keypad (on the instrument itself).  There is no password

  5. Press “F4” to allow for PC control

  6. Open UV-probe

  7. Press connect

  8. Set the wavelength range, scan speed, and interval by pressing the method button on the toolbar

  9. Load your solvent into both sample compartments in the UV-Vis and press “baseline”

  10. Press “start” to take samples

Taking Spectra

1.     To take a background, fill 2 cuvettes with the same solution, place them in the front slot (sample) and rear slot (reference), and click on Baseline

a.     The program will ask for range of wavelength. Type in 700 to 200nm, then click ok

2.   Replace the cuvette in the sample slot with your actual sample. Be sure that the cuvette is 2/3 full.

3.   Click Start

4.   After the scan is done, select where to save the data

5.   To view data points, click on the icon that looks like a paper with writing on it. (to the right of the icon with an M in a circle)

6.   To organize data, copy and paste data onto your own excel sheet

7.   To save a single scan, go to file>save as.


Turning Off UV-VIS

1.   Click Disconnect

2.   Flip switch on UV-VIS off

3.   Always turn the system off when you do not plan to use it soon to conserve the lamp life


Acrylamide and Agarose Gels and Stains

Purpose

This SOP covers the procedure for Protein gel electrophoresis.

Summary

• Acrylamide, ethidium bromide, and other stains used in gels are hazardous materials that can cause long term health effects.

• Casting gels can present hazards from chemical exposure and burns when heating.


• Running gels presents hazards of electrical shock.


• Gels must be disposed of properly and not in the normal trash.


What are acrylamide and agarose gels?


Gel electrophoresis is a technique used to separate mixtures of DNA, RNA, and proteins based on molecular weight and charge. By providing an electrical charge across a stationary phase, charged samples are separated as they move through a gel medium, which influences sample migration based on size and charge of the analytes and porosity of the medium. Gels are typically made of agarose or polyacrylamide of varying percentages dissolved in a buffer and solidified or polymerized, respectively. Stains are incorporated into the gel to allow for sample visualization.

Acrylamide is most commonly used in the production of polyacrylamide polymers. Apart from its wide use in the manufacturing industry, it is commonly used in laboratories for the purpose of polyacrylamide gel electrophoresis (PAGE) that is typically used to separate proteins and peptides based on electrophoretic mobility. A common stain used to detect proteins is Coomassie blue, which typically consists of a solution of Coomassie blue dye in glacial acetic acid and methanol. The dye turns from red unbound in solution to blue when it associates with proteins through non-covalent interactions. Dyes containing silver and various fluorescent tags are also often used.

Agarose is made up of polysaccharides and is commonly used in laboratories for the purpose of agarose gel electrophoresis for the separation of DNA and other polynucleotides. Aqueous solutions of ethidium bromide are commonly used in agarose gel electrophoresis for the purpose of visualizing nucleic acid bands. Ethidium bromide fluoresces orange upon exposure to UV light. It is a flat, planar, and aromatic compound capable of intercalating, or inserting between DNA base pairs (which are also flat, planar, and aromatic), and the resulting complex structure is stabilized by π stacking. Fluorescent intensity of ethidium bromide increases dramatically once intercalated versus the emission from solution phase solvated form thus enabling visualization. Though ethidium bromide is the most popular type of fluorescent intercalating stain, others such as SYBR safe are widely used as well.

What are the hazards?

Ethidium bromide and acrylamide are both hazardous chemicals as defined by the OSHA Hazard Communication Standard described in 29 CFR 1910.1200(d), and are the primary chemical safety concern when creating or working with gels in labs. Exposure can occur via inhalation (if aerosolized), ingestion, and skin absorption. Because of its ability to bind to DNA, ethidium bromide is a potent mutagen capable of causing genetic damage. Acute exposure can cause irritation to the eyes, mouth, skin, and upper respiratory tract.

The monomer acrylamide is a probable human carcinogen and may also cause adverse reproductive and nervous system health effects. While toxicity and potential for exposure substantially decrease after polymerization, exposure remains a concern as complete polymerization cannot be assured. Exposure to acrylamide can cause both chronic and acute health effects including ataxia (loss of muscle coordination), numb limbs, weakness, and drowsiness.

Physical hazards are presented during the heating portion of agarose gel preparation. Spilling and splashing may occur during the heating, mixing, and pouring of the hot liquefied gel, which poses a burning hazard to the body including the face, hands, and upper body. Electrocution is also a potential hazard as typical voltages of 100 V can be applied across gels resulting in 25 mA of current.



What Activities Could Pose a Risk?


Laboratory workers are at risk for exposure when making gels using either ethidium bromide or acrylamide. Inhalation exposure is a concern especially when working with the pure, solid forms of these chemicals, as dust can easily be dispersed throughout the air during operations such as pouring, weighing, mixing, or any such manipulations. Staining and destaining operations can also lead to solvent exposures if these are not conducted with appropriate containment (i.e., in a fume hood).

The process of heating and mixing the elements required for agarose gel electrophoresis poses a risk for splashing and burns to the body as well as dermal exposure to ethidium bromide, as it is typically pipetted into the mixture directly after heating. The flask containing the mixture is very hot and may lead to burning of the hands if proper protection is not used. Swirling the heated mixture and moving the flask close to the face when checking for complete dissolution of the agarose powder may lead to splashing of the mixture into the face, hands, arms and upper body leading to burns and dermal exposure to ethidium bromide. Pouring the hot mixture into the gel casting tray may also lead to splashing onto hands and arms.


The process of heating the mixture in a microwave and relying on the occurrence of boiling as an indicator of completion can also pose a hazard as the mixture may not appear to boil even if it is very hot and the powder fully dissolved. Once the powder is fully dissolved during the heating process, no material remains to serve as a source of nucleation for boiling, which can enable the mixture to superheat and flash boil, especially if it is moved directly after heating. If no boiling is observed, researchers might continue to heat the mixture unintentionally causing it to superheat, which can lead to flash boiling and splattering.

Running gels can also pose a potential for electrocution if the equipment is not in good working order, connected improperly, mishandled, or improperly grounded.


How Can Exposures Be Minimized?


As with any other hazardous material, always conduct a thorough risk assessment and employ the hierarchy of controls to minimize risk when working with acrylamide, ethidium bromide, or when heating agarose mixtures in the microwave. Some specific applications of the hierarchy of controls to these hazards are listed below.


Always apply the controls in the order of most effective to least effective (see graphic), and apply as many controls as possible to reduce the risk to the lowest achievable level.


Elimination/Substitution

• Replace high concentration products with low concentrations or less hazardous substitutes, whenever possible.

• Purchase premade solutions of ethidium bromide and acrylamide at desired concentrations to avoid working with the solid forms which are more concentrated and pose a greater inhalation hazard.


Engineering Controls

• Avoid inhalation by using engineering controls where possible in dispensing, mixing, and pouring areas, such as local exhaust ventilation (e.g. chemical fume hood). Work with solid ethidium bromide and acrylamide should always be conducted in areas with adequate ventilation or containment. Local enclosures and fume hoods are good options. All work with solvents for staining and destaining should be conducted in a fume hood.

• Routinely maintain local exhaust ventilation systems and check that chemical fume hoods are operating at the proper face velocity before each use.

• Ensure that laboratories are negatively with respect to the halfway.


Administrative Controls

Before use:

  • Consult the manufacturer’s Safety Data Sheet and additional chemical information at https://cems.unh.edu/umass/CEMS/SearchSDS.

  • Locate nearest eyewash and shower and confirm that they are accessible and within 50 ft. of the work area. This is particularly important when working with corrosive stains (e.g., coomassie).

  • Ensure all gel apparatus is in good working order. Do not use damaged connectors or cables that are frayed. Use power supplies that have load protections. Follow the manufacturer’s directions regarding assembly and use of gel apparatus.


• If weighing solids of ethidium bromide or acrylamide, take an aliquot from the bulk supply that closely approximates the amount needed and place in a closed container while working in a fume hood or local enclosure. It is best to tare the container prior to use and then make final weight adjustments very carefully at the balance to avoid dust formation.

• Designate and label areas in the lab where ethidium bromide is used and stored.


• Cover areas where gels are to be poured with a bench cover and change out when it becomes contaminated or is spilled on.


• Clean areas where hazardous materials are weighed, dispensed, or used on a regular basis to reduce accidental exposure.


• When heating liquids in the microwave, use boiling stones to ensure that a nucleation site is always present in heated solutions that do not have convective forces capable of initiating nucleation (i.e., constant stirring or shaking). Teflon boiling stones are chemically resistant and do not contaminate solutions:https://www.fishersci.com/shop/products/saint-gobain-chemware-ptfe-boilingstones/0919120#?keyword=teflon+boiling+stones. Adding 4-5 stones to each batch of agarose will prevent superheating and flash boiling.


• Always wait a few moments after heating liquids in a microwave before moving to lower the probability that any localized super heating will result in flash boiling.

Personal Protective Equipment

• Always wear appropriate PPE when making gels including:

Safety glasses or chemical splash goggles and face shield (when heating liquids in a microwave)

  • Lab coat and closed toed shoes o Nitrile gloves o Thermal gloves when handling hot materials

• In cases where engineering controls do not provide sufficient protection for inhalation exposure, please contact EH&S. We will work with you to determine the appropriate respirator based on a risk assessment for work conducted using acrylamide, ethidium bromide, or other hazardous materials. Respirator users must enroll in the University’s Respiratory Protection Program (https://ehs.umass.edu/respiratory- protection-program).


Waste Handling


Treat waste as hazardous and dispose of appropriately. Each of the following waste streams should be kept separate and referred for disposal through CEMS
https://cems.unh.edu/umass/CEMS/RequestRemoval:

• Contaminated debris (e.g. bench covers, pipette tips, gloves) and used gels should be bagged and placed in a labelled solid hazardous waste container. Contaminated glass should be placed in a puncture resistant container before placing in the solid waste hazardous container.

• Solid forms of ethidium bromide and acrylamide can be disposed of as hazardous waste in their original container or in a sealed, labelled bag.

• Stock solutions of ethidium bromide and acrylamide can be disposed of as hazardous waste in their original container.

• Buffers containing any amount of acrylamide or ethidium bromide should be referred for hazardous waste disposal through CEMS. Exposure and Spill Procedure In the event of a spill involving ethidium bromide, acrylamide, or other hazardous material that does not involve the contamination of a person, the material may be cleaned up if it is safe to do so following the general procedure for small spills detailed in the University’s Chemical Hygiene Plan.

• Ensure that it is safe to clean up the spill. o Spills of more than 1 L should be immediately referred to EH&S (413-545-2682) and the area should be evacuated. Do not permit entry to the area until EH&S arrives.

• Ensure that cleaning up the material will not generate airborne dust or aerosols. o Spills of highly volatile solvents or hazardous powders outside of enclosures (fume hoods or other enclosure devices) should be immediately referred to EH&S (413-54-2682) and the area should be evacuated. Do not permit entry to the area until EH&S arrives.

• Use absorbent material to contain and remove the liquid. After the liquid is removed rinse the area with water and contain and remove with absorbent material.

• Place all items used for clean-up in a labelled hazardous waste container and request a pickup through CEMS.


• If at any point you are uncomfortable cleaning up the spill or require assistance, stop and call EH&S (413-545-2682). Exposures to hazardous materials should follow the general procedures for exposures outlined in the University’s Chemical Hygiene Plan. For a major exposure requiring the use of a drench shower or eyewash:

• Have someone call 911 (report the building name, room number, and street address) or 413- 545-3111 (or simply 5-3111 from a campus line) to report the incident and request medical help. If possible, communicate to the first responders the nature of the exposure (e.g. ethidium bromide, acrylamide, solvents, corrosives, hot materials).

• Help the affected individual to position their head over the eyewash and activate it, or position them under the drench shower and activate it as appropriate. o Always ensure your own safety before helping others. Only help if it is safe for you to do so. o Wear gloves, safety glasses, and a lab coat.


• If using an eyewash: Instruct the affected individual to open their eyes and roll them around while the water is flowing. Help them to hold their eyes open if necessary and safe to doso. • If using a drench shower: Remove all clothing from the affected area while under the shower. • Flush the affected area for 15 minutes with water.


Risk Assessment


The overall health and safety risk for use of this material in accordance with the procedure and protocol in the following section is considered LOW based on:

  • The total quantity of any potentially hazardous material handled/transferred any one time is small – milliliter quantities or less.

  • Personnel wear protective clothing to prevent skin contact from splashes and for proper clean up practices.

The primary hazard is a spill or splash from improper or poor handling practices.

Gel Documentation System

v  Purpose

To provide instruction for the proper use of the gel documentation system (GelDoc).

 

v  Scope

All users of the gel documentation system should be familiar with the procedures outlined below.

 

v  Safety Precautions

Ethidium bromide is used as a fluorescent dye in molecular biology laboratories. It acts by inserting itself between nucleic nucleic acid strands (intercalates) and acts as a frameshift mutagen.

Always wear a lab coat, goggles and gloves when handling ethidium bromide solutions and stained agarose gels.

UV light is harmful to the skin and eyes. Do not look into the UV light source without face or eye protection.

 

v  Description of the Task

Preparation:

·           Transport the stained gel in a sealed rigid container, so that it can be carried without gloves

·           It is also permissible to use the "one-glove" technique:

o   Remove the glove from your dominant hand. Use the ungloved hand to open all doors and carry the gel (placed in a secondary container) in the gloved hand.

o   Open the door to the exposure chamber with the ungloved hand. Place the gel into the exposure chamber using the gloved hand and a spatula.

o   Use the ungloved hand to manipulate all controls on the GelDoc (procedure below)

o   Sign the log book with the ungloved hand.

o   When documentation is complete, open the chamber door with the ungloved hand and remove the gel with the gloved hand and a spatula.

o   Using the gloved hand, wipe the glass surface of the GelDoc with a Kimwipe

o   DO NOT use a paper towel as over time this scratches the glass

o   Use the gloved hand to carry the gel back to your laboratory, opening all doors with the ungloved hand.

Procedure:

Ensure you are wearing a lab coat, closed toed shoes, gloves and eye protection.

1.      Launch the software:

o    Ensuring you are using an ungloved hand, click the mouse to activate the monitor.

o    Open the GelDoc software if it is not already open.

o    On the menu bar, select ‘File’, toggle down to ‘Acquire’ and select ‘GelDoc’.

 

2.       Positioning the gel:

o    Open the chamber door with the ungloved hand and with the gloved hand load the gel into the chamber. Centre the gel, using the monitor to assist in visualization.

o    Close door and switch on the UV light.

o    With an ungloved hand, adjust the focus, zoom and aperture on the camera to obtain the optimal image.

o    In the GelDoc window, click ‘Capture’. Select the hatched-box icon in this window and drag it to select the area of interest.

o    On the menu bar, select ‘Edit’ and cursor down to ‘Extract’. A new window will appear with the final picture. You may wish to adjust the image properties such as brightness and contrast.

 

3.      Printing:

o    On the menu bar, select ‘File’, cursor down to ‘Video Print’ and click to print.

o    If the paper roll has a pink stripe, obtain a new roll from the Chemistry Stockroom and install as per the directions on the printer.

o    Close the windows containing the extracted and original images, Click ‘Don’t Save’ in the pop-up dialog box.

 

4.      Closing the program:

o    Turn off the UV light.

o    With a gloved hand, remove your gel from the chamber and wipe down the glass surface of the GelDoc with a Kimwipe.

o    Close the door to the chamber with the ungloved hand.

o    Record your name and the number of photos taken in the log book.

 

Equipment Malfunction:

If the software fails to launch, try restarting the computer. Quite often after power outages or brown outs, the system fails to launch properly.

 

Incident Response:

If EtBr solution comes in contact with your skin, wash the area thoroughly with soap and water. Inflammation and discolouration of the skin may occur after contact.

If EtBr solution comes in contact with your eye, irrigate immediately at the designated eye wash station. Flush for 20 minutes into open eye.

Complete the Incident Report Form to report the spill or incident to your supervisor..

 

Spill Cleanup:

If the gel is dropped or the staining solution was spilled:

·         Cordon off the area of the spill. Ask for assistance from those nearby if necessary.

·         Ensure you are wearing a lab coat, closed toed shoes, gloves and eye protection.

o    If the spill occurred on benchkote, remove the affected section and place in a leak proof bag or waste container identified for EtBr waste.

o    If the spill occurred on a hard surface:

§  Absorb any liquid/gel with paper towels or absorbent pads.

§  Place used materials in a leak proof bag or waste container identified for EtBr waste.

§  Decontaminate spill area as described below.

 

If EtBr powder is spilled:

·         Avoid raising dust when cleaning solid spills by mixing with water and then absorbing the solution. All spill cleanup materials and absorbents should be placed in EtBr designated waste bins. Follow with decontamination as described below.

 

Decontamination:

To decontaminate the area, it is recommended that a solution of sodium nitrite and hypophosphorus acid be used. Add 4.2g sodium nitrite and 20 mL of hypophosphorus acid to 300mL of water. Alternatively, a 10% bleach solution can be used.

·      Soak a paper towel with the decontamination solution and wash the affected area.

·      Use a UV-light to locate any remaining EtBr.

·      Use water and paper towel to clean area again. Rinse the area a few times to be sure any residue has been cleaned up.

·      Place all used material in the EtBr waste and submit a Chemical Waste Disposal Request form

 

v  Waste Management and Environmental Responsibility

Place used materials (gels, paper towels, benchkote) in waste container lined with a leak proof bag and labelled as EtBr waste. Dispose of waste by sealing bag and tagging with yellow hazardous waste disposal tag. Complete Chemical Waste Disposal Request and submit to EHS.

Buffer solutions can be collected in appropriate containers and tagged for disposal as hazardous waste, or filter the solution using commercially available filter or activated charcoal filtration. Filtrate may then be disposed of down the sink. Spent filters to be disposed of as hazardous waste.

HPLC

1.  System Start-up

Ø       HPLC

1)      Turn on HPLC from the left side as well as on the front panel.

2)      Turn on computer and login with given password.

3)      Open “LabSolutions” (icon on computer desktop) to run the HPLC system from the computer.

4)      Login with ID and password.

5)      Click “Run samples” icon on a new pop-up window.

6)      Select project (Select an existing project or create a new project)

7)      Select HPLC system.

8)      Click “OK.” The system set-up (“Connecting to LabSolutions’) will take a few minutes.


2.  Solvent Phase Transitioning

Ø       It may be necessary to transition the HPLC system from one solvent system to another. Transitioning may be column specific so consult the column’s user manual before attempting any of the following procedures. If you do not correctly transition a solvent system you could destroy lakhs of rupees worth of equipment so please make sure you understand what you are doing and that you do it correctly.

Ø       In general, you should transition between normal and reverse phase solvent systems by choosing an intermediate solvent that is miscible with both systems, purge the system, and gradually introduce new eluent(s). Note that you should label and separate solvents that are used for eluent and washing to prevent cross contamination.

Ø       Here is an example protocol for moving between an H2O/MeCN system to an iPrOH/Hex system:

a)  Disconnect the initial column and seal each end. Proper column storing envolves noting, by way of a label on the column, the solvent composition and date at which the column is being stored (ex. Jan 1, 2019 Stored in 90% H2O/MeCN)

b)  Attach both solvent inlets to an MeOH intermediate washing solution and run the system for10-20 column volumes, ensuring that you also flush the injector loop several times (do this by leaving it in the inject position for several loop volumes of solvent, and/or injecting several loop volumes of the transition solvent)

c)  Place both solvent inlets and place them in a volume of washing iPrOH and run for 5-10 column volumes, also being sure to flush the injector loop

d)  Attach solvent line “A” and “B” to your eluent solvents (iPrOH/Hex) and run the system through with 5-10 column volumes with each solvent. Be sure to flush the injector loop again.

e)  Attach column and equilibrate to the solvent composition at which the column was stored.

Ø       Refer to the column transition chart for further details and specific procedures.


3.  Purge the HPLC pumps under the following conditions:

-  starting the pumps for the first time after it has been idle for more than 24 hours.

-  after changing solvents.

The system must be purjed to remove air bubbles and for the pump to function properly.


4.  Configure the LabSolutions software to run your method.

1)      Click “Develop method” icon on the window of LabSolutions program.

2)      Click “Create New” icon.

3)      Click “PCM” ICON

4)      Set up the flow, event, and solvent settings

Ø       Flow Settings:

a.       Set pump mode to Isocratic or Gradient

b.       The table that appears below the isocratic of gradient option box is where you will enter your elution method.

*If using a gradient elution method, it is required that you include a short column washing sequence at the end of your analytical gradient. This will prevent unwanted bands from being retained on the column and getting undesirably eluted in the next run.


5)      Save this Instrument Method with a new name.


6)      Under “Develop Method,” select “New Method.”

Ø       Set your desired instrument method.

Ø       Automated data processing and reporting options can be configured here as well. Consult the Water’s LabSolutions2 Manual if you wish to do this (not recommended for small data sets).

Ø       Save this Method Set with the same name as your Instrument Method.


7)      In the main LabSolutions window enter the following into the appropriate fields:

a.       Sample name: put your sample number/ID. It is advised your first injection of the day be a blank sample to obtain a spectrum background.

b.      Function: Inject samples.

c.       Method set: select your method set which you created following above the procedure

d.      Injection volume: doesn’t matter.

e.       Run time: same total time as your Instrument Method


5.  Preparation of a sample for analytical HPLC

1)      Prepare a ~50 µM solution using HPLC solvent or other appropriate solvent

2)      If the sample or solvents have not be previously HPLC chromatographed, filter the sample solution through a 0.2 um filter. You may not inject unfiltered samples.

3)      Use volatile buffers ONLY. Addition of salt to the instrument will eventually cause precipitation, and additional maintenance. Consult the manuals.


6.  Injection

1)      Click “monitor”, and wait for the detector to show a steady, flat base-line and the HPLC pressure reading to become steady. If this does not happen, there is a problem with the system and someone from the Garner lab should be consulted. Double check connections for leaks.

2)      After getting the background result, click “monitor” to watch the flat baseline for 2-4 min on the screen.

3)      Once the baseline is flat, it’s time to inject your sample following the same procedure for the background injection. Make sure to enter your sample ID instead of “blank”


7.  Washing

Ø       After using HPLC, wash the column and injector systems. This procedure will vary depending on the nature of samples you were running. The procedure below is general for samples were mixtures of normal small molecule organic compounds. If your sample may contain compounds that are unusual or contained metals, a special cleaning procedure may be required.

Ø       If using aqueous buffers, remove all bases, buffer salts, or acids by washing with the washing solution (10% MeOH/H2O). Clean the injector by filling the sample loop with the washing solution and turning the port from load to inject (this is “flushing the injector”). If the buffers contained phosphate, leave any phosphate contaminated pump on 0.05 mL/min with pure water overnight. Try to avoid using phosphates at all costs.

Ø       If using normal phase solvent, flush the injector with iPrOH while washing the column with 100% iPrOH

Ø     If using MeOH or MeCN in water, flush the injector with MeOH while washing the column with 100% of MeOH or MeCN.


8.  Shut-down

Disconnect HPLC and power off from the left

Shutdown the PC

FTIR

  • Sign on the logbook.

  • Turn on the computer and sign in.

  • Tune on the power of the instrument, wait until the initialization succeeded. In the small panel of instrument “Perkin Elmer Spectrum 100 Series” will be shown if the initialization succeeded, otherwise come to see Jianhua first.

  • Clean the sample holder by acetone with Kimwipes, make sure not to splash the acetone on the instrument.

  • Launch the “spectrum” software on the desktop with User Name of “Analyst” and Password “analyst”, then select “Spectrum 100”.

  • Select “Instrument setup” button, “Scan and Instrument Setup” dialog will pop up, input your sample name, scan range (the range limit is 650-4500 cm-1), and scan number.

  • Click the “back ground” button to collect back group information of the sample holder.

  • Place you sample on the sample holder. If your sample is liquid, you can go ahead and press “Apply” and then “Start” to collect the spectrum. If your sample is solid, click the “monitor” button on the “Scan and Instrument Setup” dialog first, and then lower down the pressure arm, through the “monitor” dialog to monitor the total pressure applied to the sample, set the “Force Gauge” to be around 80, then click “finish”. Press “Apply” and then “Start” to collect the spectrum.

  • Data processing:

  • Peak label: Select “view” from the menu, and then “label peaks”. If you want to label a special peak, “view”-------“cursor”--------------------------------------------- “vertical continuous”, then move
  • the cursor to the peak you want, and then “view”-------- “label cursor”.
  • Data saving: you can save your data as ASCII by “File”------------ “Save as”, the
  • select “ASCII (*. ASC) as the save as type.
  • Turn off the software, log off the computer, and switch off the instrument. Make sure to clean the sample holder completely before you leave.

  • No corrosive chemical, such as strong acid and base, is allowed to be placed on the sample holder. The corrosive sample will do permanent damage to the sample holder, and you will response for the total cost of the repair.

  • Clean the sample holder and take away your samples and trash after each time usage. Failure to do that will lead to your privilege of using the instrument to be suspended.

Standard PCR Thermal Cyclers

Purpose

These instruments carry out Polymerase Chain Reaction (PCR) to amplify nucleic acid molecules. This process requires the cycling of temperatures from ~4°C - 95°C in the reaction chamber. In addition, the qPCR machine uses a diode array and detector to quantify and monitor the PCR reaction.

The PRIMARY HAZARD is the risk of burns from contact with the heating block. Take care when loading and unloading samples to prevent minor contact burns.


Basic Training Requirements

Lab personnel must have attended an instrument specific training session with the Biochemistry Instrument TA or Instrument facility approved manager prior to any use, covering general use and safe practices of the instrument in question.


Personal Protective Equipment (PPE)

No Additional PPE is required beyond what is stipulated by the General Use Safety Policy.


Respiratory Protection

None required


Hand Protection

None required unless by the demands of a users personal experiment.


Eye Protection

Standard Goggles.


Skin and body Protection

Lab coat, long pants, closed-toed shoes.


Hygiene Measures

Avoid touching instrument surfaces with gloved hands. Wipe instruments with 20% EtOH – dampened towel following use.

Never Touch Computer surfaces (mouse, keyboard etc) with gloved hands.


Procedure

  • Remove any hand protection being worn.

  • Initialize instrument and computer (for qPCR) as demonstrated during training.

  • Set qPCR or PCR program.

  • Load samples into heating block

  • *You may wear hand protection if demanded by experiment, remove before Step 5.
  • Securely close the qPCR or PCR door or lid, respectively, and begin cycle.

  • At the end of the cycle, open the PCR system.

  • Carefully remove samples and avoid extended contact with the heating block.

  • *You may wear hand protection if demanded by experiment, remove before Step 8.

  • Close down the instrument, and computer if necessary.

  • Wipe instrument handles and with towel dampened with 20% EtOH, and computer if necessary and soiled during experiment.


Ultrasonic Cleaner

  • Make sure the surrounding work space is clean and dry.

  • Never immerse the cleaner in water.

  • Unplug the cleaner while filling it with solution.

  • Do not operate dry.

  • Maintain proper fluid level.

  • Monitor  the fluid’s temperature. Never let it exceed 70C.

  • Do not use solvents, high vapor pressure or other flammable materials.

  • Keep hands out of the tank while it is operating.

  • Do not place objects on tank.

  • Use only water-based solutions.

  • Do not use mineral acids.

  • Change solutions regularly.

Cleaning Principles

An ultrasonic cleaner relies on a process called cavitation to gently and thoroughly clean the surface of your parts. An ultrasonic transducer attached to the cleaning tank transmits high frequency (47 kHz) sounds into the cleaning solution. During the low pressure stage of each sound wave tiny bubbles form and grow. During the high pressure stage these bubbles collapse, removing dirt, rust and other contaminants. This cleaning action can reach any exposed surface, even into deep crevices.


Preparation

·         Make sure the work space is clean and dry.

·         Make sure the tank and cleaning solution are clean.

·         If starting with a new solution select one appropriate for the job. See the table at the end of this procedure for assistance.

·         If cleaning will be done using the indirect method, i.e. placing the items in a beaker or solid tray and placing the beaker or tray into the tank, then the cleaner should be added to the tray or beaker. Water can be used to fill the tank.

·         Unplug the power cable.

·         Fill the cleaning tank to the proper level. Allow for the volume of the parts to be cleaned.

·         Plug in the power cable.

·         Turn on the power and wait for the start-up self-test to complete.


Degass

Degassing the cleaning solution will improve cleaning efficiency by removing dissolved gas from the cleaning solution. Degassing is usually done the first time a new solution is used or if the solution in the cleaner has not been used for a couple of days.

·         Select “Set Degass”.

·         Set degass time for 5 - 10 minutes.

·         Press ON/OFF to start.

·         Wait for the degass operation to finish.


Cleaning

·         Select “SET TEMP” and set the tank temperature. Cleaning is generally faster at higher temperatures.

·         Select “SET SONICS” and set the cleaning time.

·         Place items into a basket, tray, or in beakers. Do not place the items in direct contact with the tank. This could damage the transducer.

·         Cover  the  tank  using  the vented cover.          This will increase heating rate and will reduce evaporation of the cleaning solution.

·         Press ON/OFF to start.

·         Monitor the cleaner during operation. Do not let the cleaning solution drop below the correct level. Monitor the temperature. (Select “SOLUTION TEMP”)

·         The cleaner will stop automatically after running for the specified time.


Finishing Up

·         Remove all items from the tank.

·         Rinse, dry and/or lubricate items immediately.

·         Allow the solution to cool to a temperature where it can be handled safely.

·         Unplug the power cable.

·         Pour the used solution out, either into the drain if appropriate, or else into a proper waste disposal unit.

·         Dry the tank and the area around the cleaner.


Solution Types

Acidic Water-Based Solutions: remove rust, tarnish or scale. They range from mild solutions that remove tarnish to concentrated, inhibited acidic solutions that remove investment plaster, milk stone, zinc oxide and rust from steel and cast iron as well as smut and heat treat scale from hardened steel.


Alkaline Water-Based Solutions: include carbonates, silicates and caustics. These cause emulsifying action on the cleaned surface which keeps soil from redepositing on the cleaned surface, and improves cleaning action in hard water.


Caustic Solutions: used to remove rust from steels, metal alloy corrosion, and a variety of tenacious soils.

Autoclav

An autoclave is a large vessel containing steam under high pressure. It mainly used to sterilize different materials in the laboratory. As itoperate at high temperature and pressure, it disinfects material from microorganisms and spores contamination.

Autoclave heating up the material at a specific temperature for a specific period of time. It is also termed as steam sterilizers or steam autoclave. It is based on the moist heat sterilization method, it considered the most reliable and effective method of sterilization among all physical methods.


Uses of Autoclave


Autoclaves provide a physical method for disinfection and sterilization. They work with a combination of steam, pressure and time.


  1. They are used to decontaminate certain biological waste and sterilize media, instruments and lab ware.

  2. Autoclaves are used to sterilize medical equipment, glassware, surgical equipment, and medical wastes.

  3. Regulated medical waste that might contain bacteria, viruses and other biological material are recommended to be inactivated by autoclaving before disposal.

  4. Autoclaves are used for the sterilization of culture media, autoclavable containers, plastic tubes, and pipette tips.

Steps of Autoclave sterelization

1.     Put everything that needs to be sterilized inside the chamber and lock it.

2.     A vacuum pump sucks the air out of the chamber. The steam entering the chamber – and the pressure it creates – also work to remove the air.

3.     The autoclave sterilization temperature is crucial. Make sure it has enough time to heat up.

4.     The steam continuously covers whatever is in the autoclave, effectively killing bacteria and microbes.

5.     At the designated time, the steam is released and the chamber can be opened. The freshly-sterilized materials can be removed and they’re ready for use or disposal

Precautions

Although autoclaves are pretty simple to use, there must be some precautions to be followed while operating an autoclave.

1. 

Autoclaves
should not be used to sterilize water-proof or water-resistant substances like oil or powders.

2.     The autoclave should not be overcrowded, and the materials should be loaded in a way that ensures sufficient penetration of articles by the steam.

3.     The items to be autoclaved should always be placed in a secondary container.

4.     Only autoclavable bags are to be used to autoclave packaged waste.

5.     To ensure sufficient penetration, articles should be wrapped in something that allows penetration by steam, and materials like aluminium foils should not be used.

6.     The items placed inside the chamber should not touch the sides or top of the chamber.

7.     The wastes and clean items should be autoclaved separately.

8.     Never open the lid when the autoclave is working.

9.     Liquid components should never be autoclaved in sealed containers.

10.  The liquid inside the containers should only be filled 2/3rd of the total volume to prevent the spilling of the liquid.

11.  Plastic or polyethylene trays or containers should not be used as they might melt and damage the autoclave.

12. Besides, never autoclave flammable, reactive, corrosive, toxic or radioactive materials, household bleach, or paraffin-embedded tissue.

13. The paper should not be placed directly inside an autoclave as it is a combustible substance. It should be autoclaved in a waste bag on a bio bag setting to prevent fire.

Journal Club/ Seminar

  • Journal Club/ Seminar is held on every Monday, Tuesday, Wednesday and Saturday from 10.00 a.m. to 11.00 a.m. (During Covid-19, Journal Club/ Seminar is held on every Tuesday from 11.00 a.m. to 12.00 p.m.)

  • Seminar in charge prepares schedule for Journal Club/ Seminar.

  • All PhD and M.D. students of the Department are involved in this academic activity.

  • Senior resident act as moderator ofJournal Club/ Seminar.

  • Research papers are presented in Journal Club and subject specific topics are presented in Seminar.

  • Presenter is asked to make presentation as per the guidelines.

  • Presentation should be completed in 40 minutes so that there is enough time for discussion by all the Faculty members and students of the department.

  • Topics presented are entered in the register and it is signed by faculty members attending the same.

Seminar Library

  • Books are kept in book shelf in the seminar room.

  • List of the books available in the seminar is regularly updated.

  • Books are organized and taken care off by the seminar in charge regularly.

  • Books issued are entered in the issue register along with date.

  • Once the issued books are returned, name of the faculty member is striped off and the date is noted down.