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CLEVELAND STATE UNIVERSITY

Laboratory Equipment

Manual Stilwell Hall, Room 402

Moo-Yeal Lee

1/29/2014

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Table of Contents

Autoclave…………………………………………………………………………………………3

Balance……………………………………………………………………………………………6

Biological Safety Cabinet………………………………………………………………………..8

Cell Counter…………………………………………………………………………………….11

Centrifuge…………………………………………………………………………………….…17

Deionized Water System………………………………………………………………………..21

Hot Plate………………………………………………………………………………………...23

Incubators, Gas Tanks, Regulators, and Change-Over System……………………………..26

Microscope………………………………………………………………………………………34

Sonicator………………………………………………………………………………………...37

Vortex Mixer………………………………………………………………………………...….40

Water Bath……………………………………………………………………………………...42

Update History………………………………………………………………………………….44

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Steris Amsco Lab 250 LV 250 Laboratory Steam Sterilizer

(Autoclave)

Purpose: The autoclave is used to sterilize nonvolatile solid containers (glass) and nonvolatile

liquids (H2O and PBS) by application of high temperatures and pressures to thermally degrade

bacteria and mold without evaporating water.

Materials: Appropriately sized autoclave bags, materials wished to be autoclaved, containers for

liquid that is wished to be autoclave, autoclave.

Note: The autoclave is in room 403 of Stilwell/Fenn Hall. It belongs to Dr. Belovich’s group.

Ask her students first before using the autoclave. They may have their own particular use for it,

so be respectful of their work first before yours in that room and with that machine. Of the

pictures below, the autoclave’s door and chamber are closed on the left and open on the right.

Prepping Materials to be autoclaved:

For solids:

1. Make sure your solids are dry. Place solids in the blue autoclave bag. You can fill the bag

about ½ to ¾ full.

2. Remove the adhesive white label on top and fold over onto the blue opening of the bag to

seal closed. It’s okay if the bag folds, but you do not want the fold to occur in the region

of the adhesion, or your chance of getting contamination in the bag increases greatly

because there is effectively a hole in the sterile area. If this should happen, remove the

materials from the bag and start with a new bag.

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For liquids:

1. Fill liquids in a bottle that can be autoclaved to desired volume, but no more than the

maximum volume that can be measured by the vessel.

2. Place the autoclave tape on the outside of the bottle, both on the cap and on the bottle

itself.

Using the autoclave:

1. Log into the autoclave using the keypad screen above and on the right side of the

autoclave chamber by entering the default username and password.

2. Put items into an autoclave pan in room 403, and close the coor.

3. Autoclave materials:

a. For solids, use the gravity cycle under gravity sterilization procedures. Generally, use

a 30 minute “wet” cycle and a 30 minute “dry” cycle.

b. For liquids, use the liquid cycle under gravity sterilization procedures. Adjust the time

as necessary depending on the volume (greater volumes means more time in the

autoclave). For 1 L solution, 30 minutes at 121oC is sufficient.

4. It will take five minutes for the autoclave to start up. When the procedure is done, wait

for the autoclave to cool down before removing materials. Cap liquids on the lab bench

once the autoclaving is done. Clean up any spills that might have occurred

5. If the autoclave is to be used again within the hour, keep the door closed. If not, put the

autoclave into standby mode.

6. In case of an emergency, shut down power box on wall.

Additional Safety Precautions and Tips:

Use some sort of indicator to let yourself and others know what materials have and have

not been autoclaved. For liquids, this is a striped tape that darkens after being autoclaved.

For solids, our blue bags we use for autoclaving will have a green bar and a yellow circle

on the back of them that will darken when autoclaved.

Do not over pack the bags when autoclaving solids as you want the moisture to escape

when you autoclave the material. If you need to autoclave a lot of materials, it is better to

use several more loosely packed bags instead of a few tightly packed ones.

Additionally, do not over pack the autoclave itself for the same reasons you don’t want to

over pack the bags. You want steam to circulate and to leave your solid materials dry.

All bottles should be loosely capped (the cap should be able to move around without a

seal created between the cap and the top of the bottle), including autoclaved liquids. You

can also autoclave the caps and bottles separately if liquid is not in them. However,

tightly capping bottles can build up pressure and potentially explode in the

autoclave!

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Liquids and solids cannot be autoclaved at the same time except for the containers

holding the liquid.

Plastics that can be autoclaved include polycarbonate, teflon (PTFE), silicone, and

polypropylene. Make sure that your plastic can be autoclaved by checking material

properties first, or you risk melting and deforming the materials, and possibly damaging

the inside of the autoclave. Glass can be autoclaved.

You cannot autoclave volatile liquids or you risk explosion of the autoclave.

Always wear gloves when placing items into the autoclave. If removing autoclaved

materials, you need thermally protective gloves on if you intend to remove items from the

autoclave, or you can burn yourself. Additionally wear goggles when removing items

from the autoclave.

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Precision Balance

Purpose: The balance is used to measure exact weights of materials.

Operation:

1. Ensure that balance is placed on a flat sturdy surface, check power cord and supply

2. In order to obtain accurate measurements, it is necessary to place weighing

paper/equipment on balance. With the door closed, press the Tare button to reset the

balance to 0. This helps ensure that only the sample is being measured not the equipment.

3. The weighing paper can then be loaded with the sample to be weighed.

4. All measurements should only be taken with samples placed at the center of the balance

and with the doors closed.

5. To prevent any contamination it is necessary to clean the balance regularly (especially

before and after usage). Mild disinfectants may be used for this purpose (see figure

below).

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Biological safety cabinet

Purpose: The primary purpose of a biosafety cabinet is to serve as a means to protect the

laboratory worker and the surrounding environment from pathogens. The biosafety cabinet is an

enclosed, ventilated laboratory workspace for safely working with materials contaminated

with pathogens requiring a defined biosafety level.

Figure 1. Optimum height to which the cabinet can be left open when in use

Starting up with biosafety cabinet:

1. Open the sash (glass door) of the biosafety cabinet by pushing up on the gray handles

with both hands gently. The door should be open no higher than when the bottom surface

of the sash is at the level of the word “sash height”, but should be open enough to allow

arms to move around freely (see figure above).

2. Immediately after opening, using a kimwipe or a paper towel, wipe down the interior

surfaces of the cabinet with 10% bleach, followed with 70% ethanol to prevent pitting of

the stainless steel, and allow drying. Wait 15 minutes with the cabinet open before

beginning experiments.

3. When working in the biosafety cabinet, use proper aseptic technique. This includes

wearing gloves and a labcoat. Anything that enters the biosafety cabinet (including

yourself) should be sprayed with 70% ethanol immediately before placing inside the

cabinet.

4. Segregate all clean and contaminated materials in the work area and arrange materials to

minimize the movement of contaminated materials into clean areas. Avoid moving

materials or excessive motion of the operator’s hands and arms through the front access

opening during use.

5. If there is a spill or splatter during use, all objects in the cabinet should be surface

decontaminated before removal. Thoroughly disinfect the working area of the cabinet

while it is still in operation. This will prevent the release of contaminants from the

cabinet.

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Figure 2. Components of the biosafety cabinet

Post usage:

1. Spray 70% ethanol and wipe the surface.

2. Turn the UV light 'ON'. Close the cabinet and let the light stand for 5 mins.

3. If the UV light doesn’t turn off automatically, turn it off.

Personal and environmental safety:

Turn off UV when not in use to minimize exposure and increase the life of the light.

Ensure that the sash is set in the correct operating position.

Make sure the light and cabinet blower is on when in use. The light might need to be

turned off for some experiments, but for proper asceptic technique, always ensure that the

blower is on.

Check the return air grilles for obstructions, and note the pressure gauge reading.

Allow the cabinet to operate unobstructed for at least fifteen minutes before use.

Wash hands and arms thoroughly with soap.

Wear a long sleeved lab coat with knit cuffs and over-the-cuff gloves.

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Disposable contaminated objects, including gloves, should disposed properly after use.

Reusable objects in contact with contaminated material should be surface decontaminated

before removal from the cabinet.

All open trays or containers should be covered before being removed from the cabinet.

Open flames SHOULD NOT be used.

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Moxi Z Mini Automated Cell Counter from Orflo

Purpose: The Moxi Z Mini Automated Cell Counter calculates the average concentration of cells

in a given volume by counting the cells based on specified size parameters. The results include

the concentration of cells and the distribution of cell sizes.

Materials: 75 µL cells in transparent solution (sterile media, PBS), Cell counter, Moxi Z

Cassette. Your cells should be in an isotonic solution without detergent or DMSO as these

reagents can affect your counts. Also, before running a set of counts, run one calibration test

using the calibration beads (stored in the refrigerator).

Procedure:

Initial Injection

1. Wear gloves and a lab coat. Make sure the Cell Counter is plugged in and turned on.

2. Clean the initial area by spraying a kim wipe with 70% ethanol in deionized water and

wipe the area and the surface of the cell counter. Avoid spraying the cassette.

3. The command window will tell you to place the cassette into the port. Place Moxi Z

Cassette into the port of the Cell Counter.

a. Push the port opening down.

b. Slide the cassette into the counter above port.

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c. Push until securely in.

4. When you hear a short motor/rattle sound, add 75 µL cells in solution by pipetting

sample into the hole of the Moxi Z Cassette at a moderate speed. The media will be

sucked into the cassette.

5. Press the screen to initiate counting of the cells. Counting should take a few seconds (10-

20 seconds). The data will be automatically saved to the device.

6. After counting is done you can repeat steps 3-5 with the other side of the cassette.

Note: the calibration beads should have an average diameter of 10 µm with a standard deviation

of 0.703 µm, and a total concentration of 120,000 beads/mL. Use a pure 75 µL sample of beads

to minimize the error from pipetting. The sample recommended range is 3,000 - 500,000

cells/mL for mammalian cells.

Fitting the data:

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1. Sample results are displayed in histogram format after the counting is done (see below).

To obtain the data, press the “histogram” icon on the bottom of the screen. When the

histograms have loaded, click your sample’s histogram.

2. You can adjust the range of sizes present on your histogram. Press “Rescale” to change

the size of particles you want to measure, and “recalc” to reset the particle/cell diameter

to 2-34 µm (see below)

3. You can also manually manipulate the threshold for counting by touching the “curve fit

count” button (see below). Two blue bullseyes will appear at the top of the histogram.

These buttons can be manually slid with your fingers to determine cutoff ranges.

Alternatively, you can automatically generate gates by touching any desired point within

your peaks. Touching “gated count” will return the display to curve fit mode.

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4. You can also press the “zoom” icon to rescale the vertical axis of your histogram.

5. Press “delete” to erase the results of a test, or press “done” to return to the home screen

and save the results. The zoomed view will not be saved, but the changed horizontal axis

will.

Retrieving the data: When on the home screen, press the “histogram” button in the bottom left

corner. Up to 9 files can appear on the page simultaneously, but you can have 500 files stored on

the device at once. Use the “page up” and “page down” buttons at the bottom of the screen to

navigate, find, and select your files.

Before analyzing your data, you need to have the software from moxi Z installed on your

computer. The USB can be found in the manuals drawer (see below).

For a PC:

1. Insert the MoxiZ USB into a USB port on your PC. Click on “open to view files”.

2. Click on the “MoxiChart_Win_Install.exe” icon when the folder pops up.

3. When the installation wizard window pops up, click “next”.

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4. When the license agreement pops up, make sure you click “I accept the agreement.”

Then, click “next”.

5. Choose a location for your installation. You need at least 4.7 MB free space. After you

select a place for installation, click, “next”.

6. You will get the option to create and name a start menu folder. Choose your own

personal preferences. Then, click “next”.

7. You will again have the same option for a desktop icon. Choose your own personal

preferences, then click “next”.

8. When you’re ready to install, hit “install”. If not, press the “back” button to correct any

previous steps. To return to the install screen, just click “next”.

9. After completion of the installation, you will have the option to start up the software.

Choose what you will, then click “finish”.

For a Mac:

1. Insert the MoxiZ USB into a USB port on your PC. Open the folder.

2. Double click on the file “MoxiChartInstaller.dmg”.

3. Drag and drop “MoxiChart” to your applications folder.

4. From the applications folder, select “MoxiChart” to launch your application.

To Transfer your Data from the Cell counter to your computer:

1. On the homescreen of the cell counter, click the Bluetooth icon (2nd

to right icon on the

bottom of the screen).

2. On your PC, make sure your MoxiChart software is on, and click the Bluetooth icon in

the upper right corner.

3. Choose the device which corresponds to the moxi Z.

4. Select the folders to which you want to upload your files to.

You can update the software on your computer via automatic updates or from orflo.com. To

update the Cell Counter, you can do one of two things:

A. With the Bluetooth mode on for the cell counter, hit the power button rapidly four times.

If successful, text under the upstarting moxi Z logo will indicate the machine is in

firmware update mode.

B. With the Bluetooth mode on for the cell counter, go to the help menu on moxi chart, and

select “update firmware” from the “tools” menu.

Tips and Hazards

Use the cell concentration range as described previously.

Avoid having liquids come in contact with the outlet or anything but the cassette hole.

Try to avoid touching the back of the cassette when loading samples: hold it at the sides.

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Use personal protective equipment (gloves, and possibly a lab coat and goggles).

Try to make sure you keep the area by the counter sterile. Throw away any cassettes if

you suspect them being contaminated.

If the calibration beads do not correspond close to the proper range, order more

calibration beads. If the result is still wrong, call the orflo manufacturer

(www.orflo.com).

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Centrifuge (Eppendorf 5702)

Purpose: The primary function of the centrifuge is to aid in separating aqueous solutions into its

components based on density.

Controls:

Part Function

Time Start & stop button/

Time dial

Short Short run button

Speed Speed selector dial &

rpm/rcf button

Stand by button

Open Lid release, opens lid

1 Line socket with fuse

set and main power

switch

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E Emergency lid release

Operation:

1. Flip switch found on the back of the device to turn on centrifuge. The lid should open

automatically and then load rotors.

2. A maximum weight/ load capacity is indicated on each rotor. The fixed angle rotor can be

loaded with a maximum of 30 x 15ml round bottom test tubes. Swing bucket rotors can

accommodate either rectangular or round buckets.

3. It is necessary to insert adapters into the buckets to help stabilize the test tubes.

4. The total time allotted for centrifugation may be adjusted using the Time button.

Remaining time is displayed as the process occurs.

5. Speed can also be altered in increments of 100 min-1

or rcf using the speed button. The

short button can be used to conduct quick runs at maximum speed. The centrifugation

stops as soon as the button is released.

Maintenance and cleaning

Centrifuge:

Always unplug power cord of centrifuge with the lid open before cleaning both outer and

inner surfaces with mild cleaning agent. One may use ethanol, isopropanol or other

alcohol based disinfectants. Regular cleaning will aid in preventing corrosion to the

device.

Unscrew rotor (clockwise) carefully using rotor key. The rotor key should be cleaned

separately.

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The Rotor chambers must be cleaned only with damp cloth as any contact of the motor

shaft with liquids may result in excessive damage.

Thoroughly rinse rubber seals in the rotor chamber and lubricate with talc or glycerin

afterwards.

Should glass breakage occur during centrifugation gently remove all splinters from the

device..

Contact Eppendorf if any questions should arise concerning maintenance methods or

damages to the device and its accessories.

Rotors:

Buckets, rotors, adapters and tube holders should be cleaned at least once a month using

mild cleaning agents.

Do not immerse any of these accessories in disinfectants or cleaning agents. Conduct

regular inspections to determine mechanical damage, corrosion or presence of residue.

Rotors, tube holders, caps and buckets can however be autoclaved at 121°C for

20minutes when necessary.

Troubleshooting

Error/ Display Cause Solution

Red stand by button Centrifuge not ready Press standby button

No display No power

Power failure

Check power plug on both ends

Check fuse box of device under

main plug

Lid will not open Power failure

Running rotor

Press emergency lid release

Wait for motor to stop

Centrifuge will not start Lid not closed Secure lid/ press OPEN button

Lo on Centrifugation parameters

locked against adjustment

Open centrifuge lid.

Simultaneously press SHORT

and OPEN for 5 seconds to

readjust parameters

Inb Unevenly loaded rotor Load properly, repeat run

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Int Power failure Check power cord, let rotor stop

before repeating run

Er 2 Unbalanced rotor switch Contact maintenance/ service

personnel

Er 3 Error in speed system Leave device switched on. Error

message will disappear in

5minutes max.

Er 5/ LID Lid switch /Lid not locked

Lid not released

Secure lid and restart

Press emergency lid release

Er 6, 8 – 25 Drive/ electronics error Repeat run

Er 7 Over speed Problems in speed or drive

measurement

Er 23 Overheating motor Allow motor to cool down

Safety precautions:

Prior to any centrifugation, ensure that the centrifuge is placed on a sturdy flat surface

and is devoid of any objects or materials that can be destroyed within at least 30cm on

each side.

Ensure that the test tubes to be used are in acceptable condition in order to prevent

sample loss and damage to the centrifuge itself.

Inspect rotors for any signs of cracks, corrosion or mechanical damage. Do not use rotors

if any of the aforementioned occurrences are perceived. Only use extremely mild

cleaning agents to wipe rotors prior to and after usage.

Rotors must be firmly secured in place (it is pertinent that when fitting the rotors in the

grooves on the underside of the rotor are aligned with motor shaft) and must be

symmetrically loaded with identical buckets/ tubes so as to maintain mechanical stability

during centrifugation. In instances where there is an insufficient amount of sample for

adjacent tubes, one may use test tubes filled with an identical volume of a nontoxic

aqueous solution such as distilled water.

All tubes must be sealed tightly with lids prior to centrifugation. The centrifuge lid must

be closed as well before and during operation.

Do not move or knock centrifuge during centrifugation.

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PURELAB flex ELGA (Deionized Water System)

Purpose: The PURELAB flex ELGA deionized water system converts distilled tap water into

deionized water that we may eventually use for chemical and biological experiments. It also

dispenses water in fixed volumes specified by the user.

Operating Instructions:

1. Press any button to activate the unit.

2. In order to manually dispense the water press the Dispense Button until required amount

is dispensed. The pressure applied on the Dispense Button determines the volume of the

water being dispensed.

3. Pressing the Dispense Button and Accept Button simultaneously allows you to dispense

water freely without continuously pressing the Dispense Button. To stop dispensing just

press the Dispense Button once.

4. Press the AutoVolume Button to switch to Auto mode for predefined volume of water.

Press the Up and Down Button to adjust the quantity as required then press the Dispense

Button once to dispense water.

5. In order to enter the setting mode, press the Accept Button when the unit is in manual

mode(display showing 6L ).Press up and Down Button to scroll through the list of

settings and press the Accept Button to select the desired setting.

Dispense Button

AutoVolume

Button Accept Button

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6. The height of the dispenser can be adjusted by sliding the dispense head holder up and

down. Make sure to turn the knob on the bottom of dispense head holder in the desired

lock and unlock position as desired.

Important safety instructions:

Do not connect the power supply in the way where it comes into contact with the water.

Do not open the UV lamp cover in any case while the unit is activated. Failure to do so

can cause serious injury to eye and skin.

UV Chamber Purification pack

Lock/Unlock

knob

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Hotplates, stirrers and stirring hotplates

Purpose: Hot plates are used to heat and/or stir the solutions in the laboratory.

Setting the stirring speed:

1. Make sure the hotplate is clean and devoid of contaminants.

2. Take a clean beaker place it on the hotplate. The metallic stirrer is placed along the

diameter of the base of the beaker, their centre coinciding. The stir bar should be able to

lie completely flat in the center of the mixing beaker.

fig.1: position of the stirrer fig. 2: stirring solution at 120 rpm

3. Plug in hotplate and make sure the connection is grounded.

4. To set the speed, turn the STIR knob to the optimum or required rpm (rotations per

minute). The optimum rpm is usually when you see a small vortex at the liquid-vapor

interface in the center of the mixing vessel, but it will vary depending on the liquid and

the experiment.

5. Speed increases when turning the knob clockwise and decreases when turning

counterclockwise.

6. To turn it off rotate the knob in counter clockwise direction till it indicates “0”. You

should feel it click.

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Setting the temperature:

1. Follow the first three steps in the above section on stirring.

2. To turn the heat ON, turn the heat knob to the desired temperature. (NOTE: The

temperature on this system is specified in “ ”).

3. To turn the heat off, turn the knob in the counter clockwise direction till it reaches the

“OFF” position. You should feel it click.

fig.3 :Heating the solution at 100 .

4. CAUTION- HOT TOP indicator illuminates when the plate reaches temperature of

50 . When heat is turned off indicator will continue to flash until the temperature

drops below 50 .

5. In case of heating metal vessels or sand baths use lowest temperature possible. This is

safe with the system but may reduce the life of the stirring hotplate.

Avoid personal injury:

Plug in only where there is a good grounding connection.

Unplug during filling and emptying the tank.

Do not immerse the equipment in water.

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Keep the top surface clean. Acidic and alkali spills damage the top that leads to thermal

failure. Unplug the unit and remove spills promptly.

In case of a damaged top (etching, scratching or chipping), replace top immediately.

Do not use a foil or hotplate that obstructs the air flow, this leads to overheating.

Periodically check all the detachable connections to avoid fire hazards.

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CO2 cylinders and regulators, HeraCell 150i CO2 Incubator,

and Gas cylinder change-over unit GM 2

Purpose: The CO2 incubator (below) provides a 37oC ambient temperature condition within the

incubator. This allows proper mammalian cell growth, while using a feed of CO2 gas to maintain

proper media pH. The amount of gas coming into the incubator is regulated by CO2 regulators.

The gas cylinder change over unit allows for use of another CO2 gas cylinder after the first

cylinder runs out of CO2, allowing there to be a constant flux of CO2 into the incubator. These

pieces of equipment have been lumped together because they will be used together.

http://www.idealscientific.com/707/heracell-150i-co2-incubators/

Before starting work in the incubator:

Make sure both the incubator and the change-over unit are plugged in and turned on.

The incubator should be set to 37oC and 5% CO2.

The gas cylinders should be on (with the regulators on them), and set to an outlet pressure

of no more than 100 kPa (indicated by a red dash on the outlet (left) pressure gauge, see

below).

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There should be an adequate water level within the incubator, and the incubator should be

sterile (see sterilization procedures below).

The gas line coming out of the change-over unit should be equipped with a gas permeable

0.2 µm (or smaller) filter to ensure sterile conditions.

The biggest risk to the incubator is contamination. Try to practice aseptic techniques. Should

the incubator or the safety cabinet become contaminated, this affects all experiments that use

either of those devices. When working with the incubator (for experiments or for

decontamination):

Always where a lab coat and nitrile gloves.

Always spray your gloves with 70% ethanol in deioinized water before placing your

hands in the incubator.

Anything that goes into the incubator must also be sprayed with 70% ethanol in

deionizied water.

Minimize the duration for which the incubator door is open. Properly fasten the glass

door and close the outside door after something is removed from or placed into the

incubator.

Decontaminating the incubator must be performed before any experiment is to be started up. If

you are starting an experiment while the incubator is already in use, this does not need to be

performed. Decontamination of the incubator must also be performed as a troubleshooting

measure to make sure your cell lines are not contaminated. To decontaminate the incubator:

1. Make sure all samples and experiments are removed from the incubator.

2. Make sure the incubator is on (Plugged in and switch). The plug is white and is located

behind the incubator. The switch is in the front of the incubator at the bottom left leg

beneath the door. When on, the switch turns bright green and lights up (see below).

3. Remove the copper shelving units from the incubator (see below for image of inside the

incubator).

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4. Remove all water at the base of the incubator by pumping out water or wiping with a

cloth.

5. Spray a kimwipe with 70% ethanol in deionized water and wipe down all inside surfaces

of the incubator, including both sides of the glass door. Alternatively, you can lightly

spray the inside of the incubator with the ethanol, but you must avoid spraying the mesh

structure on the roof of the incubator. It contains the CO2 sensor, and spraying that

particular part of the instrument can permanently effect the ability to maintain the correct

pCO2 in the incubator.

6. Spray all surfaces of the shelving unit with 70% ethanol in deionized water and wipe dry

with a kim wipe. Place shelving unit back into the incubator immediately after it is dry.

7. Place 350 mL deionized water back into the incubator.

8. Close and seal both incubator doors (see below).

9. Activate the decontamination routine by pressing the “contra-con” button on the menu,

followed by “start” (see below). Decontamination will last for 25 hours.

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During decontamination, the incubator will be at temperatures in excess of 90oC! Do not open

the incubator! You could seriously burn yourself or those around you, not to mention forcing a

restart of the whole procedure. Additionally, avoid coming into direct contact with the incubator

as there could be heat exuded from the incubator during this time.

After the decontamination is done, remove the remaining water and add two liters of deionized

water to the reservoir at the bottom of the incubator (see below). Wait for the temperature and

gas levels to adjust before starting experiments.

When the incubator is not being used for experiments, make sure the gas cylinders are closed, the

incubator is closed and shut off, and the incubator (white power cord), and the change-over unit

(black power cord) are both unplugged. This saves energy and cost of resources.

Important parts of the gas cylinder regulator:

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Changing the gas cylinder tanks:

1. Close the valve on top of the gas cylinder tank by turning it clockwise. If the pressure is

too high, wait a few minutes for the pressure to drop down at the outlet. If the gas tank is

empty, the inside pressure should read zero.

2. Once the pressure has stabilized, use an appropriate sized wrench to loosen the

connecting piece between the regulator and the tank. To loosen the regulator connecting

piece, force the wrench counterclockwise. If it starts to become loose, you can manually

turn the piece counterclockwise until it comes off. Set the regulator down at the closest

table.

3. Remove the tag from the gas cap and record the information on the tag. Cap the gas tank

with the appropriate lid by lightly screwing on (see below for gas cap image). Give the

tag to Becky Laird. Leave the gas cap on the tank and put an “empty” label on the tank

until a new one is delivered. Leave the tank chained to the wall.

4. When a new gas cylinder arrives, please exchange the tank with the gas cylinder

company.

5. Chain the gas cylinder onto a structurally stable wall.

6. Unscrew the gas cap and save for when the gas tank needs to be returned. Do not remove

the tag from the cap.

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7. Screw the regulator connecting piece and the regulator back onto the new gas cylinder by

manually turning clockwise. When it because too difficult, continue turning clockwise

with an appropriately sized wrench until you feel some tension from the wrench. You

need enough pressure for there to be a seal on the regulator, but too much pressure can

also strip the regulator.

8. To test to see if the regulator is properly on, SLIGHTLY turn the open/close valve

towards the open direction.

a. Normal flow sounds like leaked flow but a bit more muted. You should hear flow

going through the tube, and it should almost sound contained, like a noise in

another room.

b. If there is a small leak, (without adjusting) feel around the edges of the regulator

to see if it’s coming from where the regulator is screwed on to the tank. Close the

tank. Take off the regulator and put it back on again. If the leak persists again,

close the tank and notify another lab member, or contact the gas company to let

them know of a faulty tank. A small leak will sound like a small leak coming out

of an air mattress or a floatation device.

c. If there is a big leak, close the tank it immediately, and wait at least several

minutes. Then, take off the regulator and put it on again. If you continue to hear a

leak, contact the gas company. A large leak will be readily apparent and carry a

great volume.

To operate the gas cylinder change over unit:

Make sure your gas lines are not leaking and fitted into the side of the unit: both cylinder

lines should be connected to the parts marked “A” and “B” with a third line for outflow

connected into the incubator (see below).

Do not get the change over unit wet or the inside of the gas lines wet. You can either

short out the instrument to prevent it from working, or wet the filter in line with the

incubator, which is not liquid compatible.

Make sure the change over unit gas lines are connected to the cylinder(s) and the

incubator.

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1. When you PLUG IN THE INCUBATOR, make sure the plug in the changeover unit to

turn it on. The “power” light should be green when on.

2. As stated earlier, make sure your gas outlet pressure for both cylinders is between 90 and

100 kPa.

3. Press the blue cylinder buttons (see above) to select the cylinder from which you would

like to obtain gas. The left of the two lights above the cylinder button will turn yellow to

indicate that it has been selected for use. You will not be allowed to press the other

cylinder button for five seconds.

4. If the outlet pressure drops below 60 kPa, the changeover unit will automatically switch

to the other tank. It is therefore important to make sure (at least daily) that the pressure

coming out of the cylinder is within range. If the pressure reaches below 60 kPa in a

cylinder, the right light above the cylinder’s matching blue button will shine red.

5. The change over unit will begin to alarm at this time. Press the yellow button beneath the

power light to stop the alarm.

6. If the low pressure cylinder that is not being used regains pressure to above 70 kPa, the

red light will be replaced by a flashing yellow light, but the cylinder will not be utilized

by the machine.

7. If both cylinders should become empty (below 60 kPa for more than a minute), the

changover unit will continuously alarm.

Other Safety Precautions:

Do not use open flames in or around the incubator as the changes in CO2 level, the

elevated temperature, and the potential present of ethanol aerosol can cause ignition.

Do not touch power cords if you or your gloves are wet.

Do not stick your head in the incubator without some kind of gas mask on.

33

Gas cylinders that are not in use must be closed with their caps on. Any individual tank

must be chained to a supportive structure.

Gas cylinders can be transported within a room without a gas cylinder cart. However, if a

gas cylinder is to be transported to another room on campus, you need to have a gas

cylinder cart. This cart should have a base to rest the cylinder on, and a chain to hold it in

place when it is moved.

Gas cylinders should not be turned open unless there is a regulator for them.

No regulator is universal! Make sure that the regulator you are using is good for the gas

and size of tank you plan on using it to regulate. A N2 regulator for a large tank should

not be used for a CO2 tank of the same size, or for a smaller N2 tank.

Do not breathe in the CO2.

The changeover unit is highly electrical, so avoid spilling fluids on the unit.

Do not knock over any of the above equipment, including but not limited to the

incubator, the cylinders, the regulators, and the change over unit.

The alarm on the changeover unit can become quite loud, so take caution.

34

Microscope (Evo, Life Technologies)

Purpose: The purpose of this microscope is to observe cells which cannot be seen by the naked

eye. This microscope will mostly be used for regularly checking the maintenance of cells.

35

Key:

1. Power input jack- plug in adapter into jack

2. Power switch- turns device on/off

3. USB/ DVI port- for flash drives and or projectors

4. Coarse stage positioning knobs- helps hold samples/ specimens within field view

5. Stage x axis knobs- used to make left to right movements for proper positioning of

specimen

6. X axis stage brake- holds stage while images are being captured

7. Stage y axis- used to make front to back movements for proper positioning of specimen

8. Y axis stage brake- holds y axis stage while images are being captured

9. Focusing knobs- helps focus on sample

10. Objective selection wheel- used in adjusting magnifications

11. Light cube selection lever- changes light channels

12. Phase annuli selector- make sure phase annuli selector corresponds with objective for

phase observations

13. Condenser slider slot- sliders improves image quality

Operation:

1. Plug in and turn on microscope.

2. Adjust magnification with the objective selection wheel and light cube lever until active

channel on the screen reflects the applicable light cube (e.g. transmitted for ‘brightfield’

operations).

3. To improve image quality, use appropriate condenser slider.

4. Use the light on button to start illumination.

5. Focus the sample using the focusing knobs. It is important that the knob does not come

in contact with the sample at any time.

6. Adjust illumination as needed, press capture to acquire image.

7. Captured images may be magnified using the zoom feature/scale bar. Double click

specific part on image to enlarge and right clicking the image will return it to its original

size.

8. The time lapse feature may assist in obtaining multiple images within a time frame.

Secure the sample by tightening the brakes after focusing. Select the time lapse tool from

the toolbar, then select interval. One may also preset the frequency of images taken

(interval text field) and the total duration (duration text field) for which images are to be

captured.

9. The microscope is also equipped with a cell count tool present in the toolbar. A

maximum of 6 labels may be used. To tag items use the radial button next to each label.

Tags may be switched, moved or even deleted.

10. Storage of files can be made using a USB device. Images can also be stored in folders on

the microscope itself. The Quick save function in the settings dialog box, allows for

storing multiple images under an identical file name.

36

Maintenance and cleaning:

Turn off power supply prior to any cleaning and maintenance with the exception of

installing updates.

Inspect equipment for any scratches especially on the lens. Wipe LCD display with non

alcohol based cleaner. Paper towels may be used to wipe the other working surfaces. No

UV sterilization may be employed.

In the event that the LED light cubes need to be replaced, adjust the stage to allow for

easy access and loosen thumbscrew to remove the cover. Replace light cube and

thumbscrew as well.

Occasionally, software updates may be required. Download updates from

lifetechnologies website. Click on update found in the service tab under settings.

37

Ultrasonic Cleaners

Purpose: The purpose of the ultrasonic cleaner is a method to remove lipids and particles within

glass tubes by applying thermal and kinetic energy to the system via a water bath and sonication.

Operating the cleaner:

1. Depending on the volume you want to clean select the amount and type of detergent. Fill

the tank with deionized water.

2. Add the cleaning solution to the water.

3. Plug into a grounded outlet.

Fig 1. Displays the Max and Min level and the tray.

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Fig2. Clean the sonicator before and after use.

Cleaning items:

1. Set the amount of time you want to clean the items for by adjusting the time using the

arrows beneath the machine.

2. Place the items in a basket or the beakers in the positioning cover.

3. In case of beakers, add the cleaning solution to the beakers, enough to cover the items.

4. Slowly lower the basket or the beaker into the tank.

5. Turn on the sonicator and leave for the desired time

6. When the items are clean, remove them from the cleaner.

7. Rinse the clean items with clean water, dry them if necessary.

8. Unplug and clean the equipment.

Safety precautions:

1. Avoid electric shocks:

Plug in only where there is a good grounding connection.

Unplug during filling and emptying the tank.

Do not immerse the equipment in water.

2. Avoid personal injury and property damage:

Do not use flammable solvents such as alcohol, gasoline in order to avoid

fire of explosion.

The equipment or the detergent can be hot, don't make direct contact.

39

Always maintain the temperature below 70'C.

3. Avoid equipment damage:

Make sure the tank is filled with water between the 'maximum' and

'minimum' levels marked.

Do not use strong acids of alkaline solutions.

Use water soluble detergents.

The work piece should never come directly in contact with the cleaner,

hence use the supporting rack.

The cleaning detergent should be replaced time to time to avoid dirt

deposits and degrading the equipment functioning.

40

VORTEX MIXER (Fisher Scientific)

Purpose: The vortex mixer is used to rapidly mix liquids of small volumes (less than 25 mL).

Operating Instructions:

1. Make sure the 3-way rocker switch is in the Off position (center).

2. Plug in the power cord into a properly grounded outlet.

3. Push the rocker switch to On position (right) for continuous operation.

4. Push the rocker switch to Auto position (left) for intermittent/touch operation.

5. Turn the speed knob to the “1”" position. In the On position, the head immediately starts

to be in motion whereas in the Auto position, the vessel must be pushed down on the

head attachment to gain motion.

Vortex Head

41

6. Vary the speed, using the speed knob in order to get the desired mix and/or vary the angle

of contact and pressure against the head.

7. Continuous, “On” mode is for larger accessories. Intermittent, “Auto” mode is for short

mixing times of one minute or less at full speed.

Important safety instructions:

Avoid wetting or unnecessary exposures to fumes.

Do not use a cleaning agent or solvent on the front panel which is abrasive or harmful to

plastics, nor one which is flammable.

Make sure the power cord is unplugged prior to any cleaning and after the work is done.

Do not run the unit above the speed setting above “5” in continuous, “on” mode.

In intermittent, “Auto” mode a rest period of two minutes is required to minimize the

overheating of motor.

42

Isotemp Water Bath (Fisher Scientific)

Purpose: The water bath is used to heat small quantities of solution to a desired temperature by

use of heat transfer from water at moderately high temperatures.

Operating Instructions:

1. Add required amount of deionized water to bath. The water level should be nearly 3/4th

of the GREEN STAND for both the bigger and smaller vessel.

2. Set the power switch to ON position. The unit goes through a self-test which takes

several seconds and the display will show the unit's capacity in liters.

3. To set the temperature set point, turn Safety controller knob fully clockwise and press

and hold the Set button while simultaneously pressing the Increase or Decrease button.

Green Stand

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4. Heat the vessel in water bath until required. Micro-centrifuge tube needs heating for 3-5

minutes, 15ml tube needs heating for nearly 15 minutes and bigger tubes need heating for

nearly 30 minutes.

5. In order to calibrate the water bath, press the “Menu” button until display shows CAL.

Then press and hold the Set button while simultaneously pressing the “Increase” or

“Decrease” button to set the desired temperature offset.

Important Safety Instructions:

Do not use the unit in the presence of flammable or combustible materials as this device

contains components which ignite such materials and cause fire.

Avoid contact to the interior of the unit as it can reach temperatures that can cause burns.

Safety controller

knob

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Update History

Equipment Original Author Date Added Updater Date Updated

Autoclave Alex Roth 01/22/2014 Alex Roth 01/22/2014

Balance Grace Apapa 01/22/2014 Alex Roth 01/22/2014

Biosafety

Cabinet Akshata Datar 01/28/2014 Alex Roth 01/29/2014

Cell Counter Alex Roth 01/22/2014 Alex Roth 01/22/2014

Centrifuge Grace Apapa 01/22/2014 Alex Roth 01/22/2014

Deionized

Water System Pranav Joshi 01/22/2014 Alex Roth 01/22/2014

Hot Plate Akshata Datar 01/22/2014 Alex Roth 01/22/2014

Incubator et.

al. Alex Roth 01/22/2014 Alex Roth 01/22/2014

Microscope Grace Apapa 01/22/2014 Alex Roth 01/22/2014

Sonicator Akshata Datar 01/22/2014 Alex Roth 01/22/2014

Vortex Mixer Pranav Joshi 01/22/2014 Alex Roth 01/22/2014

Water Bath Pranav Joshi 01/22/2014 Alex Roth 01/22/2014