ANSC 3405 Laboratory 1

 

Title

Introduction to laboratory methods, cell biology,

hematology, and ACUC training

 

Objectives

  • To learn laboratory techniques, use of the hematocrit, prepare a blood differential slide, microscope use, leukocyte counting, and to examine cell membrane permeability in a erythrocyte fragility test
  • To become trained in Animal Care and Use at Texas Tech University so that students can help care and handle the laboratory animals

 

 

Background information

·              After class, review Chapter 2 of your textbook

 

·              Blood has been collected from 4 pigs.

o       A recently 1 week old piglet

o       A nursery piglet (3-5 weeks old)

o       A finishing pig (4-6 months old)

o       A sow (2-3 years old)

 

The sample you receive will only have a treatment number on it, therefore, you will not know which treatment your blood came from. Record the treatment number in your lab book.

 

The blood is considered “whole” because an anticoagulant called heparin has been added, therefore the platelets in the blood cannot combine with protein clotting factors to form clots. Observe the blood that was collected without an anticoagulant.

 

Practice Pipette Use

Follow the instructor’s instructions on how to properly pipette. Write down the important points to properly use the micropipette, serological pipette and pasture pipette.  Accuracy and reliability are extremely important in pipetting, especially small volumes.

Accuracy is the ability to pipette an exact volume.  If you intend to deliver 10 µL, you may actually pipette 9.9 µL.  This means your assay results will be 1% in error due to human error.  If you pipette 9.0 µL when you intended to pipette 10.0 µL, then the human error is 10%.  The accuracy of pipetting can easily be validated, as we will do.

Reliability is the ability to repeat exactly the same way each time.  If you consistently pipette 9.9 µL instead of 10 µL, then your assays will consistently run under a standard.  We use different methods to assess both accuracy and reliability.

Exercise 1: Pipetting

Pipette 1000 µL onto a tared scale.  Read the weight as accurately as possible.  Repeat this 10 times.

Calculate the mean.  How this value differs from 1000 mg (or 1 g) is the relative accuracy.  The closer to 1 g, the greater the accuracy will be.

Calculate the standard deviation (sd).  The higher the standard deviation, the less reliable was the pipetting (sd is just one measure of reliability). 

From the sd and mead, calculate the coefficient of variation as:

 

                                    Mean

CV, % = 100    X       -------

                                      SD

 

The CV is our measure of relative variation (variation relative to the mean) and our measure of reliability.

Enter the values in your lab book.  Discuss briefly how you interpret the results.

 

 

Exercise 2: Prepare a blood differential smear

 

Whole Leukocyte and Erythrocyte counts and differentials

 

A. Prepare a whole blood smear after the instructor demonstrates. List the basic steps in your lab book and key features of a proper blood smear. Use the figures below as your guide. You may try as many slides as you like until you feel you have made a good slide.

 

 

F. Fixing and staining:

o       Dip dried slide 5 times for one second each in the Hema Fixative 1. Allow excess to drain.

o       Dip the slide into the Hema fixative II solution 3-5 times (red), allow excess to drain.

o       Dip the slide into the Hema fixative I solution 3-5 times. Allow excess to drain, then rinse with deionized water. Allow the slide to dry before viewing under oil immersion and light microscope.

 

The red solution often referred to as “eosin.” The red protein binds to the “acidic” parts of the cells. The blue solution binds to the “basic” parts of the cell.

 

G. After identifying and counting 100 leukocytes on your slide, draw a diagram of each cell and label it’s features. Include why the cells are named the way they are. (example: mononuclear cells have one whole nucleus).

 

Write down the differential for your treatment in your lab notebook.

 

Exercise 3: Counting Leukocytes using the Unopette system

           

A. Prepare the “unopette” system (after TA demonstrates) and list the steps in your lab book. Draw a diagram of the hemocytometer with the cells in your lab book, noting parts that you are not sure what they are. Also document the counts for your sample.

 

 

 

Exercise 4: Prepare a hematocrit

 

A. List the main steps in performing a hematocrit. After you run your sample, draw a diagram of your hematocrit with the percentages of plasma and blood.

 

 

 

Exercise 5: Plasma collection and Red blood cell fragility test

 

 A.  Centrifuge the blood for 15 minutes. During this time, you may refer to Step one directions and count your differential and/or unopette cells.

B. Once the blood is centrifuged, draw a diagram of the plasma, “buffy” coat and erythrocytes, and estimate the percentages of each.

C. A sample that has no anticoagulant has also been centrifuged. Diagram this and label the parts.

D.  Collecting plasma/serum and performing a red blood cell fragility test

 

1. Using a pasture pipette, collect the clear liquid and place it in a small vial and label it. Do not disturb the thin white layer above the red blood cells. This can be frozen and used later to evaluate chemicals in the blood (i.e. Hormones).

 

2. Take 0.5 mL of the red blood cells and add it to 0.05 mL of the Phosphate Buffered Saline, 1 X Solution which has. 0.137M Sodium Chloride, 0.0027M Potassium Chloride, and 0.0119M Phosphates. Invert the tube gently.

 

3. Label 4 glass test tubes, #1-5. In test tube # 1, place 0.5 ml (500µL) of water. In # 2, 1 mL of water. In #3, 2 mL. #4 4 mL.

 

5. In test tube # 1, add 500 µL of red blood cells. Invert the tube 3 times, then place in front of the card with the red line. Collect the time in seconds (up to 3 minutes) of how long it takes before you see the red line through your tube.

 

Time to see red line, s

Water amount, µL

 

500

 

1000

 

1500

 

2000

6. Repeat the above step (always using 500 µL of rbc), recording the times it takes to see the red line, when distilled water is added in different volumes. Record your data in a table format as seen below:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7. Using the water amount on the x-axis and time on the y axis, plot the data on a graph. Calculate the correlation coefficient (R2) and equation in EXCEL. You can perform this in EXCEL and add the appropriate tread line to get the equation and R-square. [Insert > Trendline > Options > Equation and R2]

 

You can either redraw this graph in your notebook or, preferably, print out and attach two graphs (one for your records, one for the carbon copy) into your book.

 

 

Animal Care and Use Committee Training

 

Dr. McGlone will give a lecture on Animal Care and Use. Any person working with animals at Texas Tech are required to be certified with the ACUC. During the lecture, write down the major topics or areas examined by the ACUC.

 

After the lecture, you will be given a protocol for a prospective experiment that has not had an ACUC form submitted. Go to:  http://www.depts.ttu.edu/acuc/forms.php and download the document for the “Protocol For The Use Of Live Animals For Research, Teaching Or Demonstration (MS Word Format)”. Using the information, fill out the form, print it and bring it to lab the following week, and turn it in with your lab reports.

 

Lab questions

  1. What is the difference between plasma and serum? Why would some assays require serum rather than plasma?
  2. Based on age of the pig, what is your hypothesis for the differentials counts, WBC counts, hematocrit and red blood cell fragility test?(example, Hypothesis: Neutrophil percentages in whole will decrease with age).
  3. Email your results to lindsey.hulbert@ttu.edu by Thursday at 12:30. When you receive the class data on Friday, record all of the treatments and data in your book, using a table format. (The data will be given back in an unorganized fashion, so it is up to you to organize it in a simple table). If someone had the same treatment as yours, calculate the average percentage for that measure. Make sure you include: Treatment number (age of pig), Differential counts (one column each for L, N, M, E, B %), WBC count, Hematocrit (%blood, % plasma), RBC test with each dilution, R2 value, and equation.

 

  1. The rbc fragility test used lysis by osmosis. Describe the mechanism of how the lysis of the rbc occurred just by adding water. What features of the cell may allow it to be more resistant to lysis?
  2. After comparing the results from the class, do you think that your hypothesis for each measure was correct? Why?
  3. What would be some reasons for variations among the data? (Think about the animal, the experimenter and problems that can occur in the assays).
  4. What are some major points to consider when handling animals that will be used for demonstrations, teaching or research?

 

Conclusions

In a paragraph or two, write down some conclusions for all of the demonstrations or experiments. Include any problems that you encountered and how they would be resolved. Also include how this material helped you understand major concepts from the text and lecture. Finally, include some thoughtful questions that you still may have but did not have time to ask, or that you think other students would benefit from thinking about.