Purpose 

The purpose of this lab was to become more familiar with serological pipettes. As a group, we practiced transferring different amounts of water using serological pipettes and then weighing the water to see how accurately the pipettes were calibrated. We then performed the same thing using P100 and P1000 pipette. We then began the Ludox protocol already designed and worked on it until the end of class. Another purpose of this lab was to try and find a more effective method of Ludox gradient centrifugation that allows us to see the organic layer easiest and isolate it.

Procedure

Pipetting 

• With a serological pipette, place 1mL of water in a weighing dish and record mass
• Repeat step 1 but this time place 0.1mL of water in the dish
• Repeat step 1 but this time place 0.01mL of water in the dish
• Repeat steps 1-3 but use P100 or P1000 pipette

Ludox Protocol 

• Put 5g of top soil and 10mL  of water in a jar, and then mix for 5-10 minutes using a vortex
• Allow the soil to settle by letting the sample sit for about 2 minutes and then transfer 3.68mL of the soil water to a clean glass tube
• Add 368 microliters of 25% glutaraldehyde, and vortex the tube several times for about 30 seconds
• Add 16mL of Ludox to a 50mL conical tube Inject 4mL of the fixed sample into the Ludox using a P1000 pipette and inject it right below the surface
• Add a layer of colored water on top of the Ludox and prevent mixing
• Label and weigh the tube, then centrifuge at 4300 x gravity in a swinging bucket rotor for 15 minutes
• Remove the liquid from the cell layer and place it in 2 separate microfuge tubes using a P1000 pipette

Data and Observations 

Pipette Observations 

The mass of the 1mL and 0.1mL drops weighed exactly 1g and 0.1 respectively with both types of pipettes  0.01mL drops were to small for the scale to pick up  Ludox Observations  The protocol proved successful in producing a visible cell layer  We were able to remove it successfully and place it in 2 separate microfuge tubes  We did not have enough time to finish the predetermined protocol

Concial tube data 

Labeled: 21-1 TCA

Weight: 40.9 grams

Tube was stored in tube rack at the back of the laboratory

Microfuge tube data 

Both labeled : 21-1 TCA

Weight: 3.94 grams and 4.2 grams

Stored in rack and placed in refrigerator for examination

Conclusion 

After placing the organic layer in 2 separate microfuge tubes, we will be able to re-examine the tubes in the following lab. Due to limited time, we were not able to finish the protocol, so next week will will start with the process of isolating the pellet. The protocol we determined proved very successful in the fact that we had a great separation of cell layer.