Difference between revisions of "Team:elan vital korea/Protocol"

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<body>
 
<body>
     <!-- Section #1 -->
+
     <!-- Section #1 --!>
 
         <section id="home" data-speed="10" data-type="background">
 
         <section id="home" data-speed="10" data-type="background">
            <a name="top" id="top"></a>
+
  <a name="top" id="top"></a>
            <a href="#">
+
        <a href="#"><img class="igem" src="https://static.igem.org/mediawiki/2015/7/70/IGEM_Logo.gif"></a>
                <img class="igem" src="https://static.igem.org/mediawiki/2015/7/70/IGEM_Logo.gif">
+
            <br>
            </a>
+
            <br>
                        <br><br><br><br><br><br><br>
+
            <br>
                    <div class="center;">
+
            <br>
            <a href="https://2015.igem.org/Team:Elan_Vital_Korea">
+
            <br>
                <img class="displayed" src="https://static.igem.org/mediawiki/2015/d/d5/ElanVital_Logo.png">
+
            <br>
            </a>
+
            <br>
 +
<div class="center;">
 +
                <a href="https://2015.igem.org/Team:Elan_Vital_Korea">
 +
                    <img class="displayed" src="https://static.igem.org/mediawiki/2015/d/d5/ElanVital_Logo.png">
 +
                </a>
 
                 <h4 style="text-align:center;">
 
                 <h4 style="text-align:center;">
 
                     WETLAB <br> -Protocol-
 
                     WETLAB <br> -Protocol-
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     <!-- Section #2 -->
+
     <!-- Section #2 --!>
 
         <section id="about" data-speed="10" data-type="background">
 
         <section id="about" data-speed="10" data-type="background">
            <h5 style="text-align:center;">
+
    <h5 style="text-align:center;">
                <a name="myAnchor" id="myAnchor"></a>
+
<a name="myAnchor" id="myAnchor"></a><br><br><br>Protocols</h5>
 
<br>
 
<br>
                   
 
<font color="black">PROTOCOL</font>
 
            </h5>
 
                 
 
                    <P style="text-align:center;">
 
  
<font color="black">
 
We conducted our experiments by following the protocols below.  As an official procedure, lab workers should understand the lab experiment <br>
 
assigned to them along with safety procedures before starting lab work. The protocols are arranged according to the order of experiments we followed. <br><br>
 
                    </font>
 
                    </p> 
 
<img class="displayed" src="https://static.igem.org/mediawiki/2015/6/6e/Hr_white.jpg" width="80px" height="2px">
 
<br>
 
                </h6>
 
 
  <h5 style="text-align:center;">
 
  
 +
<div class="inner0">
 +
<P style="text-align:left;">
 
  <font color="black">
 
  <font color="black">
Protocols to handle enzymes.
+
We conducted our experiments by following the protocols below.  As an official procedure, lab workers should understand the lab experiment assigned to them along with safety procedures before starting lab work. The protocols are arranged according to the order of experiments we followed.
 
</font>
 
</font>
                    </h5>
+
</p> 
 +
</div>
  
  
 +
<h5 style="text-align:center;">
 +
 +
<font color="black">
 +
The rule of thumb is:
 +
</font>
 +
</h5>
  
 
<div class="inner">
 
<div class="inner">
  
 
  <font color="black">
 
  <font color="black">
  <div class="caption2">
 
  
 +
  <div class="caption2">
 
   <h5>
 
   <h5>
  
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     </h5>
 
     </h5>
 
         <p>
 
         <p>
Enzymes used in our project, such as AHL, must be stored <br>
+
Start with reading Safety Questions for overview.
in low temperature.The enzymes must be stored in the <br>
+
freezer when they are not used, and must be put on ice when <br>
+
taking them out of the freezer for an experiment. <br>
+
 
         </p>
 
         </p>
 
   </div>
 
   </div>
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   <h5>
 
   <h5>
  
  <font color="black"> 2 </font>
+
  <font color="black"> 2</font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
Enzymes should be added last to the solution, because <br>
+
Follow Elan Vital Safety Rules all the time.
enzymes are sensitive to inactivation by pH and <br>
+
ionic conditions that deviate from their storage and reaction <br>
+
buffers. After adding enzymes, the mixed solution should <br>
+
 
         </p>
 
         </p>
 
   </div>
 
   </div>
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</div>
 
</div>
  
 
 
 
 
 
  <h5 style="text-align:center;">
 
 
<font color="black">
 
Protocols to store materials and maintain <br>
 
usage history of each material.
 
</font>
 
    </h5>
 
  
 
<div class="inner">
 
<div class="inner">
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   <h5>
 
   <h5>
  
  <font color="black"> 1 </font>
+
  <font color="black"> 3 </font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
Reporter cell, test cell and competent cell (Top 10 invitrogen)<br>
+
Before entering the laboratory, read <br>
must be kept at 4°C and frequently used <br>
+
GMS General Safety Rules <br>
enzymes, reagents, DNA plasmids should be kept at −20°C <br>
+
and GMS Extra Rules again and follow them
in the freezer..
+
 
         </p>
 
         </p>
 
   </div>
 
   </div>
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   <h5>
 
   <h5>
  
  <font color="black">2 </font>
+
  <font color="black"> 4 </font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
We use AHL as protein enzymes. AHL must be kept at lower <br>
+
If you are unsure of the rules or confused, <br>
temperature (4°C or lower) for the recurring use. <br>
+
ask lab assistant and/or <br>
AHL can be destroyed easily when it is stored and/or handled<br>
+
instructor before doing anything.
in improper temperature.
+
 
         </p>
 
         </p>
 
   </div>
 
   </div>
 
 
</font>
 
</font>
 
</div>
 
</div>
 
+
<br>
  
  
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                   To The Top
 
                   To The Top
 
               </font> </h6>
 
               </font> </h6>
 
  
 
         </section>
 
         </section>
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     <!-- Section #3 -->
+
     <!-- Section #3 --!>
 
         <section id="maintext" data-speed="10" data-type="background">
 
         <section id="maintext" data-speed="10" data-type="background">
<br><Br>
+
<br><br>
 
+
<P style="text-align:center;">
<div class="inner2">
+
 
<font color="black">
 
<font color="black">
 +
We have conducted our experiments at Gachon University. Thus, we have always observed safety rules of the lab. We had Safety Education & Training Session <br>
 +
before starting our lab work. Also, when we conducted our experiments, Gachon University staff supervised our work.
 +
</font>
 +
</p>
 +
<br><br>
  
  <div class="caption3">
+
  <h5 style="text-align:center;">
      <h5>3</h5>
+
        <p>
+
We use triple distilled water (or <br>
+
DDH2O) to make LB broth. <br>
+
Triple distilled water is kept at lab <br>
+
temperature(around 18 °C or lower).
+
        </p>
+
  </div>
+
 
+
<div class="caption3">
+
      <h5>4</h5>
+
        <p>
+
Other materials such as yeast and <br>
+
NaCl are stored and maintained <br>
+
under the responsibility of <br>
+
Gachon Molecular  Biology Lab.
+
        </p>
+
  </div>
+
 
+
<div class="caption3">
+
      <h5>5</h5>
+
        <p>
+
We have to record the history of each <br>
+
material, including if <br>
+
plasmids/reporter cell/ test cell/ AHL have <br>
+
been frozen and if so, <br>
+
when it is used.
+
        </p>
+
  </div>
+
</font>
+
</div>
+
  <h5 style="text-align:center;">
+
 
                 <font color="black">
 
                 <font color="black">
Process
+
Gachon University General Lab Safety Rule
 
</font>
 
</font>
    </h5> <br>
+
</h5>
 
+
<br><br>
 
+
 
+
  
 
<div class="inner2">
 
<div class="inner2">
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Add 200 μL of 1000x chloramphenicol <br>
 
Add 200 μL of 1000x chloramphenicol <br>
 
and gently stir it. Be careful not <br>
 
and gently stir it. Be careful not <br>
to shake the bottle too long/hard so that <br>
+
to shake the bottle too long/hard so that
 
bubbles are created.<br>
 
bubbles are created.<br>
 
         </p>
 
         </p>
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     </h5>
 
     </h5>
 
         <p>
 
         <p>
Pour into empty petri dishes just enough <br>
+
Pour into empty petri dishes <br>
to cover the surface (~20 mL per plate).<br>
+
just enough  to cover the surface<br>
 +
(~20 mL per plate).<br>
 
In case that bubbles are in <br>
 
In case that bubbles are in <br>
the plate, heat the plate surface carefully <br>
+
the plate, heat the plate surface <br>
with a burner only until the bubbles are <br>
+
carefully with a burner only <br>
 +
until the bubbles are <br>
 
burst but the solution is heated.
 
burst but the solution is heated.
 
         </p>
 
         </p>
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     </h5>
 
     </h5>
 
         <p>
 
         <p>
Leave the plates at room temperature <br>
+
Leave the plates at room <br>
around one hour until it is solidified.
+
temperature around one hour <br>
 +
until it is solidified.
 
         </p>
 
         </p>
 
   </div>
 
   </div>
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     </h5>
 
     </h5>
 
         <p>
 
         <p>
Solidified plates should be turned upside <br>
+
Solidified plates should be <br>
down for a few hours at room temperature,<br>
+
turned upside down for a <br>
 +
few hours at room temperature,<br>
 
then stored at 4°C.
 
then stored at 4°C.
 
         </p>
 
         </p>
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</font>
 
</font>
 
</div>
 
</div>
           
+
 
 
         </section>
 
         </section>
  
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     <!-- Section #4 -->
+
     <!-- Section #4 --!>
 
         <section id="maintext2" data-speed="10" data-type="background">
 
         <section id="maintext2" data-speed="10" data-type="background">
 
<br>
 
<br>
  
  <h5 style="text-align:center;">
 
                <a name="myAnchor" id="myAnchor"></a>
 
<br>
 
  
<font color="black"> LB Medium</font>
+
 
            </h5>
+
<div class="inner3">
<P style="text-align:left;">
+
  
 
  <font color="black">
 
  <font color="black">
 +
  <div class="caption4">
  
<div class="inner">
+
  <h5>
 +
 
 +
<font color="black"> 10 </font>
 +
    </h5>
 +
        <p>
 +
Person who leaves the lab last must make sure that power is disconnected, flammable material is isolated, dangerous material is safely kept, water is disconnected, lab is cleaned and locked, and order of the lab must be double- checked.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
 
 +
<font color="black"> 11 </font>
 +
    </h5>
 +
        <p>
 +
Power wiring appliance must be properly installed to accommodate the experiments. Cables, and lines must be checked regularly to confirm that they are capable of handling the electrical load.
 +
        </p>
 +
  </div>
 +
 
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
 
 +
<font color="black"> 12 </font>
 +
    </h5>
 +
        <p>
 +
All equipment must be connected with the proper power Cable and must not be connected in a multiple power socket.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
 
 +
<font color="black"> 13 </font>
 +
    </h5>
 +
        <p>
 +
Autoclave for 20 min within 2 hr
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
 
 +
<font color="black"> 14 </font>
 +
    </h5>
 +
        <p>
 +
Keep it cool until it reaches <br>
 +
around 40-50 °C
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
 
 +
<font color="black"> 15 </font>
 +
    </h5>
 +
        <p>
 +
Add 200 μL of 1000x chloramphenicol <br>
 +
and gently stir it. Be careful not <br>
 +
to shake the bottle too long/hard so that
 +
bubbles are created.<br>
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
 
 +
<font color="black"> 16 </font>
 +
    </h5>
 +
        <p>
 +
Pour into empty petri dishes <br>
 +
just enough  to cover the surface<br>
 +
(~20 mL per plate).<br>
 +
In case that bubbles are in <br>
 +
the plate, heat the plate surface <br>
 +
carefully with a burner only <br>
 +
until the bubbles are <br>
 +
burst but the solution is heated.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
 
 +
<font color="black"> 17 </font>
 +
    </h5>
 +
        <p>
 +
Leave the plates at room <br>
 +
temperature around one hour <br>
 +
until it is solidified.
 +
        </p>
 +
  </div>
 +
</font>
 +
</div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
 
 +
<font color="black"> 18 </font>
 +
    </h5>
 +
        <p>
  
 
  <font color="black">
 
  <font color="black">
 +
Solidified plates should be <br>
 +
turned upside down for a <br>
 +
few hours at room temperature,<br>
 +
then stored at 4°C.
 +
</font>
 +
        </p>
 +
  </div>
 +
</font>
 +
</div>
 +
        </section>
 +
 +
 +
 +
 +
 +
 +
    <!-- Section #5 --!>
 +
        <section id="maintext" data-speed="10" data-type="background">
 +
<br>
 +
 +
 +
<div class="inner3">
 +
<font color="black">
 +
  <div class="caption4">
  
  <div class="caption2">
 
 
   <h5>
 
   <h5>
 +
<font color="black"> 19 </font>
 +
    </h5>
 +
        <p>
 +
Inflammable chemicals including acetone, oil, or gas must be kept in the place where ventilation is easy and access is infrequent.
 +
        </p>
 +
  </div>
  
  <font color="black"> 1 </font>
+
  <div class="caption4">
 +
  <h5>
 +
  <font color="black"> 20 </font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
We used LB medium almost every day, so we have prepared LB medium many times. To prevent contamination, we only used LB medium made within three days.
+
Importing and exporting of high-pressure gas container must be done using transportation equipment.
 
         </p>
 
         </p>
 
   </div>
 
   </div>
  
   <div class="caption2">
+
 
 +
   <div class="caption4">
 
   <h5>
 
   <h5>
 +
<font color="black"> 21 </font>
 +
    </h5>
 +
        <p>
 +
When storing high-pressure gas containers, they must be safely and firmly fixed, isolated from flammable substances and inflammatory materials.
 +
        </p>
 +
  </div>
  
  <font color="black"> 2 Materials </font>
+
  <div class="caption4">
 +
  <h5>
 +
  <font color="black"> 21 </font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
NaCl, Trypton, Yeast Extract, and ddH2O (triple distilled water)
+
Experiments using flammable, explosive, toxic, or volatile gases or vapors must be carried out in a hood.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 22 </font>
 +
    </h5>
 +
        <p>
 +
Reagents container or cabinets for storing reagents must be kept in cool, well-ventilated places without direct sunlight, and far away from fire and heat sources.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 23 </font>
 +
    </h5>
 +
        <p>
 +
Experimental drugs shall be stored in a way not to be shaken by outside shock, and so that its storage bottles do not fall
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 24 </font>
 +
    </h5>
 +
        <p>
 +
Toxic materials shall be handled using protective films, or other safety tents to prevent damages from splatter, heating or explosion so as to ensure the safety of the experimenter.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 25 </font>
 +
    </h5>
 +
        <p>
 +
Containers for chemical wastes shall not be left in the hallway or on the stairs, and shall not be stored in the corners of the laboratory or other invisible places.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 26 </font>
 +
    </h5>
 +
        <p>
 +
<font color="black">
 +
When performing microbiological tests, gloves shall be worn, and after the experiment, the used glassware shall be
 +
washed through sterilization, and disposable supplies and medium shall be disposed of separately from general garbage.
 +
</font>
 
         </p>
 
         </p>
 
   </div>
 
   </div>
Line 575: Line 789:
 
</div>
 
</div>
  
 +
        </section>
  
<div class="inner">
+
  <!-- Section #6 --!>
 +
        <section id="maintext2" data-speed="10" data-type="background">
 +
<br>
 +
 
 +
 
 +
<div class="inner4">
  
 
  <font color="black">
 
  <font color="black">
   <div class="caption2">
+
   <div class="caption3">
  
 
   <h5>
 
   <h5>
+
 
  <font color="black"> 3 Equipment </font>
+
  <font color="black"> 27 </font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
autoclave, electronic scale.
+
Inflammable chemicals including acetone, oil, or gas must be kept in the place where ventilation is easy and access is infrequent.
 
         </p>
 
         </p>
 
   </div>
 
   </div>
  
   <div class="caption2">
+
   <div class="caption3">
 
   <h5>
 
   <h5>
  
  <font color="black"> 4 Process </font>
+
  <font color="black"> 28 </font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
We usually make 200 liter LB bottle
+
Importing and exporting of high-pressure gas container must be done using transportation equipment.
1) 2 g of NaCl to a final concentration of 0.17 M
+
        </p>
2) 2g of 1%(w/v) Bacto™ tryptone
+
  </div>
3) 1g of 0.5% (w/v) yeast extract
+
 
4) ddH2O to 200 mL
+
 
5) Autoclave for 20 min within 2 hours
+
  <div class="caption3">
6) Keep at room temperature
+
  <h5>
 +
 
 +
<font color="black"> 29 </font>
 +
    </h5>
 +
        <p>
 +
When storing high-pressure gas containers, they must be safely and firmly fixed, isolated from flammable substances and inflammatory materials.
 
         </p>
 
         </p>
 
   </div>
 
   </div>
Line 608: Line 833:
 
</div>
 
</div>
  
 +
<h5 style="text-align:center;">
 +
             
 +
<font color="black">
 +
Other significant Safety Considerations.
 +
</font>
 +
</h5>
 +
<br><br>
 +
 +
<h5 style="text-align:center;">
 +
             
 +
<font color="black">
 +
1. Fire Regulations.
 +
</font>
 +
</h5>
  
  <h5 style="text-align:center;">
+
<P style="text-align:center;">
  
 
  <font color="black">
 
  <font color="black">
Overnight Cultures with Antibiotics
+
When fire breaks out, the following guidelines shall apply.
 
</font>
 
</font>
 +
</p>
 +
 +
 +
 +
<div class="inner3">
 +
 +
<font color="black">
 +
  <div class="caption6">
 +
 +
  <h5>
 +
 +
<font color="black"> 1) </font>
 
     </h5>
 
     </h5>
 +
        <p>
 +
Save lives.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption6">
 +
  <h5>
 +
 +
<font color="black"> 2) </font>
 +
    </h5>
 +
        <p>
 +
Call the fire brigade.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption6">
 +
  <h5>
 +
 +
<font color="black"> 3) </font>
 +
    </h5>
 +
        <p>
 +
Alert people in the area.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption6">
 +
  <h5>
 +
 +
<font color="black"> 4) </font>
 +
    </h5>
 +
        <p>
 +
Extinguish the fire if possible.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption6">
 +
  <h5>
 +
 +
<font color="black"> 5) </font>
 +
    </h5>
 +
        <p>
 +
Close doors to the area.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption6">
 +
  <h5>
 +
 +
<font color="black"> 6) </font>
 +
    </h5>
 +
        <p>
 +
Evacuate.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption6">
 +
  <h5>
 +
 +
<font color="black"> 7) </font>
 +
    </h5>
 +
        <p>
 +
Reassemble outside the building at the designated meeting point.
 +
        </p>
 +
  </div>
 +
</font>
 +
</div>
 +
        </section>
 +
 +
 +
 +
 +
 +
    <!-- Section #7 --!>
 +
 +
 +
        <section id="maintext" data-speed="10" data-type="background">
 +
<br>
 +
 +
<P style="text-align:center;">
 +
<font color="black">
 +
Also it is very important to know the locations of the fire-fighting equipment, fire alarms, and evacuation routes closest to the lab. <br>
 +
A small fire can be extinguished quickly by smothering it in a fire blanket or by spraying it with a fire extinguisher. <br>
 +
Lab fires in biological labs are caused most commonly by the plating of cell cultures. <br>
 +
To prevent this, <br><br>
 +
</font>
 +
</p>
 +
 +
 +
<div class="inner3">
 +
<font color="black">
 +
  <div class="caption4">
 +
 +
  <h5>
 +
<font color="black"> 1 </font>
 +
    </h5>
 +
        <p>
 +
Never use paper bench coat near burners.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 2 </font>
 +
    </h5>
 +
        <p>
 +
Use a low reservoir volume of ethanol.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 3 </font>
 +
    </h5>
 +
        <p>
 +
Place the ethanol reservoir at least a foot from the burner and on the opposite side of the burner from the plates
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 4 </font>
 +
    </h5>
 +
        <p>
 +
Never wear plastic gloves when working with a flame.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 5 </font>
 +
    </h5>
 +
        <p>
 +
Carry the gas container for the burner carefully with a firm grip. If it is dropped, make sure that there is no leakage by smelling for gas.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 6 </font>
 +
    </h5>
 +
        <p>
 +
Notify the instructor of any gas leakage and do not light burners when there is a smell of gas.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 7 </font>
 +
    </h5>
 +
        <p>
 +
Always light the match before opening the gas valve.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 8 </font>
 +
    </h5>
 +
        <p>
 +
Avoid placing burners too close to overhanging shelves.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 9 </font>
 +
    </h5>
 +
        <p>
 +
Never leave the table while a burner is on.
 +
        </p>
 +
  </div>
 +
</font>
 +
</div>
 +
 +
        </section>
 +
 +
 +
 +
 +
 +
    <!-- Section #8 --!>
 +
        <section id="maintext2" data-speed="10" data-type="background">
 +
<br>
 +
<h5 style="text-align:center;">
 +
             
 +
<font color="black">
 +
CHEMICALS
 +
</font>
 +
</h5>
 +
<br>
 +
 +
<P style="text-align:center;">
 +
 +
<font color="black">
 +
On each chemical container, there is a label that specifies the potential danger of the substance for humans and/or the environment. <br>
 +
Chemicals should be handled cautiously with gloves, both for your safety and for decreasing the contamination risk.<br>
 +
Always wear a lab coat and shoes as additional protection. Read the signs on the chemical container and the Material Safety Data Sheet<br>
 +
(MSDS; available online) for further direction.<br>
 +
</font>
 +
</p>
 +
 +
 +
<br>
 +
<h5 style="text-align:center;">
 +
<font color="black">
 +
BIO- SAFETY AND DISPOSAL
 +
</font>
 +
</h5>
 +
<br>
 +
 +
<P style="text-align:center;">
 +
 +
<font color="black">
 +
When you are working to generate new organisms by molecular methods, you are regulated in most countries <br>
 +
according to international biosafety guidelines:
 +
</font>
 +
</p>
  
  
Line 627: Line 1,102:
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
We have conducted overnight culture for a single bacterial strain which process needs a plate or medium with single  colonies and LB containing chloramphenicol.
+
CDC, Atlanta. Biosafety in Microbiological and Biomedical Laboratories. <br>
 +
<a>http://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf </a>
 
         </p>
 
         </p>
 
   </div>
 
   </div>
Line 634: Line 1,110:
 
   <h5>
 
   <h5>
  
  <font color="black"> 2 Materials </font>
+
  <font color="black"> 2</font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
chloramphenicol or ampicillin should not be added to LB before autoclaving the LB or after subsequent cooling to  room temperature for storage because antibiotics are not sufficiently stable. The solution must be added just before bacteria is added.
+
WHO, Geneva. Laboratory Biosafety Manual, 2004.<br>
 +
<a>http://www.who.int/csr/resources/publications/ biosafety/Biosafety7.pdf</a>
 
         </p>
 
         </p>
 
   </div>
 
   </div>
Line 645: Line 1,122:
  
 
<div class="inner">
 
<div class="inner">
 +
 
  <font color="black">
 
  <font color="black">
 
   <div class="caption2">
 
   <div class="caption2">
Line 650: Line 1,128:
 
   <h5>
 
   <h5>
  
  <font color="black"> 3 Equipment </font>
+
  <font color="black"> 3 </font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
Material Needed chloramphenicol: 25 μg/mL
+
ECDC, Directive 2000/54/ec of the European parliament and of the Council of 18 September 2000 on the protection of  workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC. <br>
Normal stock concentrations: 1000-fold higher
+
<a>http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2000:262:0021:0045:EN:PDF </a>
In case of using ampicillin: 100 μg/mL
+
 
         </p>
 
         </p>
 
   </div>
 
   </div>
Line 662: Line 1,139:
 
   <h5>
 
   <h5>
  
  <font color="black"> 4 Process </font>
+
  <font color="black"> 4 </font>
 
     </h5>
 
     </h5>
 
         <p>
 
         <p>
4. Process
+
Institutional Bio-safety Committee, under the ministry of Health and Welfare, Korea Center for Disease Control & Prevention. <br>
 +
<a>http://biosafety.cdc.go.kr</a>
 +
        </p>
 +
  </div>
 +
</font>
 +
</div>
 +
        </section>
  
1) Quickly burn the neck of a bottle containing LB medium before pouring it out into a tube. Even the  
+
 
slightest contamination of LB will be damaging.  
+
 
2) Add chloramphenicol or ampicillin to give the appropriate concentration
+
    <!-- Section #9 --!>
3) Scoop one colony from the plate with a sterile micropipette tip
+
        <section id="maintext" data-speed="10" data-type="background">
4) Immediately stick the tip into the tube containing the medium and chloramphenicol or ampicillin
+
<br><br><br>
5) Cover the tube loosely with aluminum foil by taping it on to allow exchange of oxygen for chemical maturation of the chromoprotein choromophores.
+
<P style="text-align:center;">
6) Incubate at 37°C with the shaking incubator overnight.
+
<font color="black">
 +
When working with microorganisms such as bacteria and viruses, there are four BioSafety Levels (BSL) numbered BSL1–4.<br>
 +
Biosafety level is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed <br>
 +
The levels of containment range from the lowest biosafety level 1 to the highest at level 4, and Ministry of Health and Welfare and Korea Centers <br>
 +
for Disease Control and Prevention have specified these levels facility. <br>
 +
Biocontainment can be classified by the relative danger to the surrounding environment as biological <br>
 +
safety levels.  As of 2006, there are four safety levels. These are called BSL1 through BSL4 <br>
 +
</font>
 +
</p>
 +
<br>
 +
 
 +
<h5 style="text-align:center;">
 +
                <font color="black">
 +
BSL 1
 +
</font>
 +
</h5>
 +
<br>
 +
 
 +
<div class="inner3">
 +
<font color="black">
 +
  <div class="caption4">
 +
 
 +
  <h5>
 +
<font color="black"> 1 </font>
 +
    </h5>
 +
        <p>
 +
involving well-characterized agents not known to consistently cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 2 </font>
 +
    </h5>
 +
        <p>
 +
bacteria and viruses including canine hepatitis, non-pathogenic Escherichia coli, as well as cell cultures and  non-infectious bacteria.
 +
        </p>
 +
  </div>
 +
 
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 3 </font>
 +
    </h5>
 +
        <p>
 +
The laboratory is not necessarily separated from the general traffic patterns in the building.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption7">
 +
  <h5>
 +
<font color="black"> 4 </font>
 +
    </h5>
 +
        <p>
 +
Conducted on open bench tops using standard microbiological practices.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption7">
 +
  <h5>
 +
<font color="black"> 5 </font>
 +
    </h5>
 +
        <p>
 +
Laboratory personnel have specific training in the procedures conducted in the laboratory.
 +
        </p>
 +
  </div>
 +
 
 +
  <div class="caption7">
 +
  <h5>
 +
<font color="black"> 6 </font>
 +
    </h5>
 +
        <p>
 +
Supervised by a scientist with proper training.
 +
        </p>
 +
  </div>
 +
 
 +
 
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 7 </font>
 +
    </h5>
 +
        <p>
 +
Contaminated materials are left in open waste receptacles.
 
         </p>
 
         </p>
 
   </div>
 
   </div>
 
</font>
 
</font>
 
</div>
 
</div>
 +
        </section>
  
 +
 +
 +
 +
    <!-- Section #9 -->
 +
        <section id="maintext2" data-speed="10" data-type="background">
 +
<br><Br>
 +
<h5 style="text-align:center;">
 +
             
 +
<font color="black">
 +
BSL 2
 +
</font>
 +
</h5>
 +
<br><br>
 +
<div class="inner2">
 +
 +
<font color="black">
 +
  <div class="caption5">
 +
 +
  <h5>
 +
 +
<font color="black"> 1 </font>
 +
    </h5>
 +
        <p>
 +
Similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption5">
 +
  <h5>
 +
 +
<font color="black"> 2 </font>
 +
    </h5>
 +
        <p>
 +
Includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting such as C. difficile, most Chalmydiae, hepatitis A,B, and C, orthopoxviruses, influenza A, Lyme disease, Salmonella, mumps, measles, scapie, MRSA, and VRSA.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption5">
 +
  <h5>
 +
 +
<font color="black"> 3 </font>
 +
    </h5>
 +
        <p>
 +
similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption3">
 +
  <h5>
 +
 +
<font color="black"> 4 </font>
 +
    </h5>
 +
        <p>
 +
Laboratory personnel have specific training in handling pathogenic agents,
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption3">
 +
  <h5>
 +
 +
<font color="black"> 5 </font>
 +
    </h5>
 +
        <p>
 +
Must be directed by scientists with advanced training.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption3">
 +
  <h5>
 +
 +
<font color="black"> 6 </font>
 +
    </h5>
 +
        <p>
 +
Extreme precautions are taken with contaminated sharp items.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
 +
<font color="black"> 7 </font>
 +
    </h5>
 +
        <p>
 +
In case that infectious aerosols or splashes may be created, specified procedures should be conducted in biological safety cabinets or other physical containment equipment.
 +
        </p>
 +
  </div>
 +
 +
</font>
 +
</div>
 
         </section>
 
         </section>
 +
 +
 +
 +
 +
 +
 +
 +
    <!-- Section #10 --!>
 +
        <section id="maintext" data-speed="10" data-type="background">
 +
<h5 style="text-align:center;">
 +
                <font color="black">
 +
BSL 3
 +
</font>
 +
</h5>
 +
<br>
 +
 +
<div class="inner3">
 +
<font color="black">
 +
  <div class="caption4">
 +
 +
  <h5>
 +
<font color="black"> 1 </font>
 +
    </h5>
 +
        <p>
 +
Indigenous or exotic agents which may cause serious or potentially lethal disease after inhalation.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 2 </font>
 +
    </h5>
 +
        <p>
 +
Includes various bacteria, parasites and viruses that can cause severe to fatal disease in humans but for which  treatments exist, such as Yersinia pestis, SARS coronavirus, Brucella, yellow fever virus.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 3 </font>
 +
    </h5>
 +
        <p>
 +
Laboratory personnel have specific training in handling pathogenic and potentially lethal agents,
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 4 </font>
 +
    </h5>
 +
        <p>
 +
Supervised by competent scientists who are experienced in working with these agents.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 5 </font>
 +
    </h5>
 +
        <p>
 +
All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets,  specially designed hoods, or other physical containment devices.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 6 </font>
 +
    </h5>
 +
        <p>
 +
The laboratory personals should wear appropriate personal protective clothing and equipment.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 7 </font>
 +
    </h5>
 +
        <p>
 +
An acceptable level of safety for the conduct of routine procedures, may be achieved in a biosafety level 2 facility, providing the filtered exhaust air from the laboratory room is discharged to the outdoors.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 8 </font>
 +
    </h5>
 +
        <p>
 +
The ventilation to the laboratory is balanced to provide directional airflow into the room.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
<font color="black"> 9 </font>
 +
    </h5>
 +
        <p>
 +
Access to the laboratory is restricted when work is in progress and the recommended Standard Microbiological  Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed.
 +
        </p>
 +
  </div>
 +
</font>
 +
</div>
 +
        </section>
 +
 +
 +
 +
    <!-- Section #11 --!>
 +
        <section id="maintext2" data-speed="10" data-type="background">
 +
<h5 style="text-align:center;">
 +
             
 +
<font color="black">
 +
BSL 4
 +
</font>
 +
</h5>
 +
<br><br>
 +
<div class="inner2">
 +
 +
<font color="black">
 +
  <div class="caption4">
 +
 +
  <h5>
 +
 +
<font color="black"> 1 </font>
 +
    </h5>
 +
        <p>
 +
Limited access only for authorized persons can work in the facilities. Not allowed for people who have weak immune system.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
 +
<font color="black"> 2 </font>
 +
    </h5>
 +
        <p>
 +
When dealing with biological hazards at this level, the use of a positive pressure personnel suit, with a segregated  mandatory.
 +
        </p>
 +
  </div>
 +
 +
 +
  <div class="caption4">
 +
  <h5>
 +
 +
<font color="black"> 3 </font>
 +
    </h5>
 +
        <p>
 +
All works should be supervised by qualified scientists who are trained and experienced in working with these agents.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
 +
<font color="black"> 4 </font>
 +
    </h5>
 +
        <p>
 +
The entrance and exit of a level 4 bio-lab will contain multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designated to destroy all traces of the biohazard.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
 +
<font color="black"> 5 </font>
 +
    </h5>
 +
        <p>
 +
Multiple airlocks are employed and are electronically secured to prevent both doors from opening at the same time.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
 +
<font color="black"> 6 </font>
 +
    </h5>
 +
        <p>
 +
All air and water service going to and coming from a biosafety level 4 will undergo similar decontamination  procedures to eliminate the possibility of an accidental release.
 +
        </p>
 +
  </div>
 +
 +
  <div class="caption4">
 +
  <h5>
 +
 +
<font color="black"> 7 </font>
 +
    </h5>
 +
        <p>
 +
Members of the laboratory staff shall have specific and thorough training in handling extremely hazardous infectious agents, and they shall understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics.
 +
        </p>
 +
  </div>
 +
 +
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Revision as of 19:33, 17 September 2015








WETLAB
-Protocol-




Protocols

We conducted our experiments by following the protocols below. As an official procedure, lab workers should understand the lab experiment assigned to them along with safety procedures before starting lab work. The protocols are arranged according to the order of experiments we followed.

The rule of thumb is:
1

Start with reading Safety Questions for overview.

2

Follow Elan Vital Safety Rules all the time.

3

Before entering the laboratory, read
GMS General Safety Rules
and GMS Extra Rules again and follow them

4

If you are unsure of the rules or confused,
ask lab assistant and/or
instructor before doing anything.


To The Top


We have conducted our experiments at Gachon University. Thus, we have always observed safety rules of the lab. We had Safety Education & Training Session
before starting our lab work. Also, when we conducted our experiments, Gachon University staff supervised our work.



Gachon University General Lab Safety Rule


1

200 mL LB prepared fresh,
non-autoclaved

2

3 g agar

3

Shake until all solids
are dissolved

4

Autoclave for 20 min within 2 hr

5

Keep it cool until it reaches
around 40-50 °C

6

Add 200 μL of 1000x chloramphenicol
and gently stir it. Be careful not
to shake the bottle too long/hard so that bubbles are created.

7

Pour into empty petri dishes
just enough to cover the surface
(~20 mL per plate).
In case that bubbles are in
the plate, heat the plate surface
carefully with a burner only
until the bubbles are
burst but the solution is heated.

8

Leave the plates at room
temperature around one hour
until it is solidified.

9

Solidified plates should be
turned upside down for a
few hours at room temperature,
then stored at 4°C.


10

Person who leaves the lab last must make sure that power is disconnected, flammable material is isolated, dangerous material is safely kept, water is disconnected, lab is cleaned and locked, and order of the lab must be double- checked.

11

Power wiring appliance must be properly installed to accommodate the experiments. Cables, and lines must be checked regularly to confirm that they are capable of handling the electrical load.

12

All equipment must be connected with the proper power Cable and must not be connected in a multiple power socket.

13

Autoclave for 20 min within 2 hr

14

Keep it cool until it reaches
around 40-50 °C

15

Add 200 μL of 1000x chloramphenicol
and gently stir it. Be careful not
to shake the bottle too long/hard so that bubbles are created.

16

Pour into empty petri dishes
just enough to cover the surface
(~20 mL per plate).
In case that bubbles are in
the plate, heat the plate surface
carefully with a burner only
until the bubbles are
burst but the solution is heated.

17

Leave the plates at room
temperature around one hour
until it is solidified.

18

Solidified plates should be
turned upside down for a
few hours at room temperature,
then stored at 4°C.


19

Inflammable chemicals including acetone, oil, or gas must be kept in the place where ventilation is easy and access is infrequent.

20

Importing and exporting of high-pressure gas container must be done using transportation equipment.

21

When storing high-pressure gas containers, they must be safely and firmly fixed, isolated from flammable substances and inflammatory materials.

21

Experiments using flammable, explosive, toxic, or volatile gases or vapors must be carried out in a hood.

22

Reagents container or cabinets for storing reagents must be kept in cool, well-ventilated places without direct sunlight, and far away from fire and heat sources.

23

Experimental drugs shall be stored in a way not to be shaken by outside shock, and so that its storage bottles do not fall

24

Toxic materials shall be handled using protective films, or other safety tents to prevent damages from splatter, heating or explosion so as to ensure the safety of the experimenter.

25

Containers for chemical wastes shall not be left in the hallway or on the stairs, and shall not be stored in the corners of the laboratory or other invisible places.

26

When performing microbiological tests, gloves shall be worn, and after the experiment, the used glassware shall be washed through sterilization, and disposable supplies and medium shall be disposed of separately from general garbage.


27

Inflammable chemicals including acetone, oil, or gas must be kept in the place where ventilation is easy and access is infrequent.

28

Importing and exporting of high-pressure gas container must be done using transportation equipment.

29

When storing high-pressure gas containers, they must be safely and firmly fixed, isolated from flammable substances and inflammatory materials.

Other significant Safety Considerations.


1. Fire Regulations.

When fire breaks out, the following guidelines shall apply.

1)

Save lives.

2)

Call the fire brigade.

3)

Alert people in the area.

4)

Extinguish the fire if possible.

5)

Close doors to the area.

6)

Evacuate.

7)

Reassemble outside the building at the designated meeting point.


Also it is very important to know the locations of the fire-fighting equipment, fire alarms, and evacuation routes closest to the lab.
A small fire can be extinguished quickly by smothering it in a fire blanket or by spraying it with a fire extinguisher.
Lab fires in biological labs are caused most commonly by the plating of cell cultures.
To prevent this,

1

Never use paper bench coat near burners.

2

Use a low reservoir volume of ethanol.

3

Place the ethanol reservoir at least a foot from the burner and on the opposite side of the burner from the plates

4

Never wear plastic gloves when working with a flame.

5

Carry the gas container for the burner carefully with a firm grip. If it is dropped, make sure that there is no leakage by smelling for gas.

6

Notify the instructor of any gas leakage and do not light burners when there is a smell of gas.

7

Always light the match before opening the gas valve.

8

Avoid placing burners too close to overhanging shelves.

9

Never leave the table while a burner is on.


CHEMICALS

On each chemical container, there is a label that specifies the potential danger of the substance for humans and/or the environment.
Chemicals should be handled cautiously with gloves, both for your safety and for decreasing the contamination risk.
Always wear a lab coat and shoes as additional protection. Read the signs on the chemical container and the Material Safety Data Sheet
(MSDS; available online) for further direction.


BIO- SAFETY AND DISPOSAL

When you are working to generate new organisms by molecular methods, you are regulated in most countries
according to international biosafety guidelines:

1

CDC, Atlanta. Biosafety in Microbiological and Biomedical Laboratories.
http://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf

2

WHO, Geneva. Laboratory Biosafety Manual, 2004.
http://www.who.int/csr/resources/publications/ biosafety/Biosafety7.pdf

3

ECDC, Directive 2000/54/ec of the European parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC.
http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2000:262:0021:0045:EN:PDF

4

Institutional Bio-safety Committee, under the ministry of Health and Welfare, Korea Center for Disease Control & Prevention.
http://biosafety.cdc.go.kr




When working with microorganisms such as bacteria and viruses, there are four BioSafety Levels (BSL) numbered BSL1–4.
Biosafety level is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed
The levels of containment range from the lowest biosafety level 1 to the highest at level 4, and Ministry of Health and Welfare and Korea Centers
for Disease Control and Prevention have specified these levels facility.
Biocontainment can be classified by the relative danger to the surrounding environment as biological
safety levels. As of 2006, there are four safety levels. These are called BSL1 through BSL4


BSL 1

1

involving well-characterized agents not known to consistently cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment.

2

bacteria and viruses including canine hepatitis, non-pathogenic Escherichia coli, as well as cell cultures and non-infectious bacteria.

3

The laboratory is not necessarily separated from the general traffic patterns in the building.

4

Conducted on open bench tops using standard microbiological practices.

5

Laboratory personnel have specific training in the procedures conducted in the laboratory.

6

Supervised by a scientist with proper training.

7

Contaminated materials are left in open waste receptacles.



BSL 2


1

Similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment.

2

Includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting such as C. difficile, most Chalmydiae, hepatitis A,B, and C, orthopoxviruses, influenza A, Lyme disease, Salmonella, mumps, measles, scapie, MRSA, and VRSA.

3

similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting.

4

Laboratory personnel have specific training in handling pathogenic agents,

5

Must be directed by scientists with advanced training.

6

Extreme precautions are taken with contaminated sharp items.

7

In case that infectious aerosols or splashes may be created, specified procedures should be conducted in biological safety cabinets or other physical containment equipment.

BSL 3

1

Indigenous or exotic agents which may cause serious or potentially lethal disease after inhalation.

2

Includes various bacteria, parasites and viruses that can cause severe to fatal disease in humans but for which treatments exist, such as Yersinia pestis, SARS coronavirus, Brucella, yellow fever virus.

3

Laboratory personnel have specific training in handling pathogenic and potentially lethal agents,

4

Supervised by competent scientists who are experienced in working with these agents.

5

All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets, specially designed hoods, or other physical containment devices.

6

The laboratory personals should wear appropriate personal protective clothing and equipment.

7

An acceptable level of safety for the conduct of routine procedures, may be achieved in a biosafety level 2 facility, providing the filtered exhaust air from the laboratory room is discharged to the outdoors.

8

The ventilation to the laboratory is balanced to provide directional airflow into the room.

9

Access to the laboratory is restricted when work is in progress and the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed.

BSL 4


1

Limited access only for authorized persons can work in the facilities. Not allowed for people who have weak immune system.

2

When dealing with biological hazards at this level, the use of a positive pressure personnel suit, with a segregated mandatory.

3

All works should be supervised by qualified scientists who are trained and experienced in working with these agents.

4

The entrance and exit of a level 4 bio-lab will contain multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designated to destroy all traces of the biohazard.

5

Multiple airlocks are employed and are electronically secured to prevent both doors from opening at the same time.

6

All air and water service going to and coming from a biosafety level 4 will undergo similar decontamination procedures to eliminate the possibility of an accidental release.

7

Members of the laboratory staff shall have specific and thorough training in handling extremely hazardous infectious agents, and they shall understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics.