1.0  Purpose:

Operation of the Bio-Tek Elx808UI Automated Microplate Reader.

2.0  Scope:

Applies to the Bio-Tek Elx808UI Automated Microplate Reader for performing optical density testing on solutions.

3.0  Responsibilities:

1. It is the responsibility of the course instructor/lab assistant to ensure that this SOP is performed as described and to update the procedure when necessary.

2. It is the responsibility of the students/technicians to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

4.0  References:

1. Bio-Tek Automated Plate Reader Operators Manual

5.0  Definitions: N/A

6.0  Precautions: N/A

7.0  Materials:

1. Samples, standards and controls to be tested

2. Micropipette.

3. 96-well microplate (U, V, or flat-bottom wells are acceptable).

8.0  Procedure:

1. Preparation

   1. Assemble samples to be tested . A minimum of 100µL for each well is required.

   2. Load samples into microplate starting at the top left corner (location A1). Load proceeding samples down the microplate, B1, C1, D1, etc. Refer to Figure 1 for a map of the microplate.

2. Operation

Turn the power switch to the ON position (located on the rear of the right-side  panel). The equipment will perform a system self-test to verify that the components are operating properly and that the internal software has not been corrupted (less than one minute).

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1.Purpose:

The purpose of this SOP is to describe the procedure in using the M Air T Millipore Air Tester in conducting airborne microbial testing.

2.Scope:

The scope of this SOP is limited to performing airborne microbial testing using the M Air T Millipore Air Tester.

3.Responsibilities:

1. It is the responsibility of the course instructor /lab assistant to ensure that this SOP is performed as directed and to update the procedure when necessary.

2. It is the responsibility of the students/technicians to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

4.References:

1. M Air T Millipore Air Tester Operation and Maintenance Instruction

2. autoclave SOP

3. incubator SOP

5.Definitions: N/A

6.Precautions: Always wear the appropriate personnel protective equipment (safety eye glasses and gloves

7.Materials:

1. M Air T Millipore Air Tester and accessories.

2. M Air T Cassette pre-filled with TSA media

3. autoclave

4. incubator

5. 70% isopropyl alcohol (IPA)

6. lab towels

8.Procedure:

8.1.Using the air tester in vertical, horizontal or inclined position

1. When using the air tester in a vertical position, the tripod is not used.

2. When using the air tester in a horizontal position or 30° from the horizontal position, the tripod is needed. Fix the tripod onto the air tester by screwing it into the tester fixing hole.

8.2.Powering up the equipment

1. If the equipment is used to collect samples inaccessible to power outlets, the equipment has internal rechargeable batteries ready for use. Press the ON/OFF button. LCD display will be turned on.

Note: Make sure the battery is fully charged. The LCD will display the battery symbol if it is fully charged.

1. If the equipment is used to collect samples accessible to power outlets, plug the power adapter into an empty and convenient power outlet. LCD display will be turned on.

8.3.Adjusting the volume to be processed

Note: Refer to Figure 2.

1. The recommended volume is 1000 Liters (1m3).

2. Setting up volume other than the recommended volume

3. To access other preset volumes, press the LITERS button multiple times until you find your desired volume.

4. To change the volume setting, select the volume that is just below the preset volume you want to process. Then hold the LITERS button until the tester display indicates the desired sampling volume.

8.4.Adjusting the timer

Note: Refer to Figure 2.

1. The recommended set time is 5 minutes.

2. Setting up the time other than the recommended set time.

   1. Hold down the START/DELAY button. The display shows preset times in increments of 5 minutes up to one hour. Select the desired time by simply releasing the START/DELAY button.

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1.     Purpose:

To perform a microbial identification assay.

2.     Scope: 

This procedure is intended as a standardized identification system for Enterobacteriaceae and non-fastidious Gram-negative rods included in the database

3.     Responsibilities:

1. It is the responsibility of the course instructor/lab assistant to ensure that this SOP is performed as described and to update the procedure when necessary.

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

4.     References:

1. API 20E System Brochure

2. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – cGMP (FDA publication, September 2004)

3. United States Pharmacopeia 25

4. Gram Stain SOP

5. Bergey’s Manual of Systematic Bacteriology

6. Oxidase Test SOP

5.     Definitions:

1. Enterobacteriaceae: Family of Gram-negative, rod bacteria that inhabit soil, water and are commonly found in the large bowel of humans. Most common organisms isolated from clinical specimens.

6.     Precautions: Aseptic technique and standard precautions for handling microbial cultures.

7.     Materials: Test Setup

1. API 20E strip, API Suspension Medium, incubator tray and cover

2. Bacterial cultures including E. coli control (ATCC #25922)

3. Disposable gloves

4. Test tube rack and sterile disposable pipettes

5. McFarland Standard # 0.5

6. Sterile wooden sticks

7. Sterile mineral oil

8. 37^oC Incubator

9. Squirt bottle - approx. 10 ml of tap water

Development

1. Oxidase Dropper, filter paper/cotton swab

2. Kovac’s (James) reagent

3. VPI and VP2 reagents

4. 10% ferric chloride

5. Nitrate reagents (I&II)

6. Zn dust

7. API 20E Result sheets

8. API 20E Quick Index Booklet or the API 20E Profile Recognition System

8.     Procedure:

1. Using the Gram-stain technique (Gram-stain SOP) determine that the bacterial culture is a Gram-negative rod.

8.2.  Oxidase test:

1. Perform the Oxidase test according to the Oxidase Test SOP.

2. Record the result on the API 20E Results sheet (21^st identification test).

8.3.  Preparation of the inoculum:

1. Open an ampule of API Suspension Medium (5 ml).

2. Using a sterile wooden stick, remove a well-isolated colony from the streak plate and transfer to the API Suspension Medium. Mix to emulsify and obtain a homogenous suspension.

Check the turbidity of the suspension to that of the McFarland Standard # 0.5. If necessary, add more bacteria.

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1.Purpose

This procedure describes the operation of the ÄKTA pure Chromatography System, controlled by Unicorn 6.3 software, for the purpose of anion exchange chromatography of samples containing green fluorescent protein (GFP).

2.Scope and Applicability

Applies to purification of GFP from diverse origins, including bacterially expressed recombinant GFP. Cell lysates may be prepared by a variety of methods. The prepared lysate sample for purification should have a pH between 7.5 and 8.2. This procedure uses a Hi Trap Capto Q HP 5 ml column installed on the GE ÄKTA pure Chromatography System and controlled by Unicorn 6.3 software.

3.Summary of Method

1. Preparation of buffer(s)

2. Equilibration of system and column

3. Fraction collector setup

4. Application of sample

5. Washing and elution of column

6. Regeneration of system in preparation for subsequent run

7. Procedures for short- or long-term storage of the system

4.References

1. Unicorn 6.3 Users Guide (electronic)

2. AKTA pure 25 Users Guide (electronic)

3. AKTA pure Operating Instructions (Note: Chapter 4 Section 5)

4. Hi Trap Capto Q HP 5 information booklet

5. SOP: Operation of AKTA Pure Chromatography System

5.Definitions: N/A

6.Precautions

1. Routine care should be exercised in handling of buffers and samples of biological materials, which may have harmful biological activity in the case of accidental ingestion, needle stick, etc.

2. User should read and be familiar with general good practice as outlined in the AKTA pure Cue Cards located near the instrument.

3. Avoid damaging the threads through the use of excessive force when connecting plastic fasteners.

4. Care must be taken to avoid air in the fluid path, which could damage the pumps or give spurious and uninterpretable readout from the UV and/or conductivity detectors.

5. Gloves and protective eyewear should be worn when handling samples and reagents (buffers), however it is preferable that the user remove gloves prior to entering commands via the computer keyboard or mouse.

6. Buffers must be degassed and filtered prior to use with the AKTA pure instrument.

7. Samples should be centrifuged at 10000xg for 5 min and then sterile filtered using a 0.2 µm filter before injection/introduction into the fluid path.

8. Equipment calibration check: The AKTA pure system calibration of A280 and conductivity are automatic; baseline for measurements of A280 and conductivity are zeroed at the beginning of a chromatography run. However, calibration of the pH detector must be performed prior to use of the instrument each day, using standard calibration buffers and the automated routine in Unicorn. It is assumed that calibration is performed according to the Equipment SOP for the AKTA pure 25 instrument. Further adjustment is beyond the scope of this document and should be referred to a qualified technician.

7.Responsibilities

1. It is the responsibility of the course instructor/lab assistant to ensure that this SOP is performed as described and to update the procedure when necessary.

2. It is the responsibility of the students/technician to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

8.Equipment and Materials

1. AKTA pure chromatography system

2. Additional Lab Equipment:  pH meter, balance

3. Lab Utensils: Beakers (250, 500ml), 500 ml graduated cylinders

4. Reagents: Tris, hydrochloric acid, sodium chloride, filtered deionized water (Milli Q or similar), 20% ethanol.

5. Lab Supplies: Filters (0.2 mm) and bottles for vacuum filtration and degassing of all chromatography buffers. Syringe (1ml). Syringe filters (0.2 mm). Tubes for fraction collector.

9.Procedure

1. Sample Collection and Preparation is described elsewhere, (for example the SOP: Preparation of Bacterial Cell Lysates using BPER Reagent DP20, 20MAY2019). This chromatography method is also appropriate for samples in compatible buffer such as 50 mM Tris-HCl, pH 8.2. The operator will require 0.6 ml of sample per sample injection (see below).

2. Reagent Preparation: Buffers should be prepared dependent on the mode of separation employed; in this instance anion exchange chromatography provides good separation of GFP from contaminating protein at pH 8.2.

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1.Purpose

Quantitative determination of total tPA

2.Scope and Applicability

Human tPA Total Antigen ELISA may be used for quantitative determination of total tPA in cell culture and tissue lysate samples as well as human plasma and other biological fluids

3.Summary of Method

1. Preparation of standard

2. Standard and unknown addition

3. Primary antibody addition

4. Secondary antibody addition

5. Substrate incubation

6. Measurement

7. Calculation of results

4.References

1. Molecular Innovations Human tPA Total Antigen ELISA Kit (Cat # HTPAKT-TOT) Manual

2. Bio Rad iMark Microplate Absorbance Reader SOP

5.Precautions: None

6.Responsibilities

1. It is the responsibility of the course Supervisior to ensure that this SOP is performed as described and to update the procedure when necessary.

2. It is the responsibility of the analyst to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

7.Equipment and Materials

1. Molecular Innovations Human tPA Total Antigen ELISA Kit (Cat # HTPAKT-TOT)

2. 20µl, 200µl, and 1000µl pipettes and tips

3. Shaking platform capable of reaching 300rpm

4. Bio Rad iMark Microplate Absorbance Reader

5. Microtubes and rack

6. Blocking Buffer (3% BSA (w/v) in TBS buffer (0.1M Tris, 0.15M NaCl, pH 7.4))

7. 1M HCl

8. 1X washing Buffer

9. tPA Samples from Spinner Flask and Bioreactor

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1.     Purpose

2.     Scope and Applicability

The Bio Rad iMark Microplate Absorbance Reader is an eight-channel, vertical path lengthphotometerthatmeasurestheabsorbanceofthecontentsinthewellsofa96-well microtitration plates. It can perform single or dual wavelength measurements and can report absorbance values to three decimal places.

3.     Summary of Method

1. Turn on and load 96 well plate into Bio Rad iMark Microplate Absorbance Reader.

2. Run protocol.

3. Remove 96 well plate and turn off Bio Rad iMark Microplate Absorbance Reader

4.     References

1. iMark Microplate Absorbance Reader Instruction Manual

5.     Precautions

1. Be sure to open and close the “Reading Chamber Door” by pressing the “Open/Close” key on the “Keypad”. Attempting to manually open or close the “Reading Chamber Door” can lead to damage.

6.     Responsibilities

1. It is the responsibility of the Supervisor to ensure that this SOP is performed as described and to update the procedure when necessary.

2. It is the responsibility of the Microbiologist to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

7.     Equipment and Materials

1. iMark Microplate Absorbance Reader

2. 96 Well Plate

8.     Procedure

1. Turn on the iMark Microplate Absorbance Reader by holding the “Power Button” until the “LCD” illuminates

   1. The “LCD” will display “Self Diagnosis” followed by “Initializing” Once initialization is complete, a login screen will appear.

   2. The login name will read “Common User” and a prompt for a password will be shown. Enter “00000” and press “Enter” on the “Keypad.”

   3. “Lab Name, Kit Name, Reading Mode, and Measurement Filter” appears on screen (be sure the Measurement Filter is set at 450nm, the filter specific for tPA ELISAs), self-initialization is complete, and the Bio Rad iMark Microplate Absorbance Reader is ready for measurement.

2. Open the “Reading Chamber Door” by pressing the “Open/Close” key on the “Keypad”.

   1. Once “Reading Chamber Door” is open, place 96 Well Plate into the “Reading Chamber” being sure to line the 96 Well Plate with the plate holding guides.

3. Close the “Reading Chamber Door” by pressing the “Open/Close” key on the “Keypad”.

4. Start measurement reading by pressing the “Start/Stop” key on the “Keypad”. The measurement function will begin and all data will be printed indicating the completion of the measurement function.

   1. Remove data printout

5. Open the “Reading Chamber Door” by pressing the “Open/Close” key on the “Keypad”.

   1. Once “Reading Chamber Door” is open, remove 96 Well Plate from the “Reading Chamber.”

6. Close the “Reading Chamber Door” by pressing the “Open/Close” key on the “Keypad”.

7. Turn off the Bio Rad iMark Microplate Absorbance Reader by holding the “Power

Button” until the “LCD” reads “Power Off” and “Yes” is highlighted, press “Enter” key. Bio Rad iMark Microplate Absorbance Reader will shut down

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