ACTIVITY 1

TITRATION OF VINEGAR

OBJECTIVES

To determine the molarity and percent by mass of acetic acid in vinegar.

INTRODUCTION:
Vinegar is essentially a solution of acetic acid in water. The concentration of acetic
acid in vinegar may be expressed as a molarity (in mol/L):

or as a mass percent

In this experiment, we will be using titration to determine the concentration of acetic

acid in vinegar. A titration involves performing a controlled reaction between a
solution of known concentration (the titrant) and a solution of unknown concentration
(the analyte). Here, the titrant is an aqueous solution of ~0.1 M sodium hydroxide
and the analyte is vinegar. When mixed, a neutralization reaction occurs between
sodium hydroxide and the acetic acid in vinegar.

The sodium hydroxide will be gradually added to the vinegar in small amounts from a
burette. A burette is a device that allows the precise delivery of a specific volume of
a solution. The NaOH will be added to the vinegar sample until all the acetic acid in
the vinegar has been exactly consumed (reacted away). At this point the reaction is
completed, and no more is required. This is called the equivalence point of the
titration. In order to know when the equivalence point is reached, an indicator
solution called phenolphthalein is added to the vinegar at the beginning of the
titration. Phenolphthalein is a pH sensitive organic dye. Phenolphthalein is colorless
in acidic solutions like vinegar, and deep pink in basic solutions like sodium
hydroxide. At the equivalence point of the titration, just one drop of will cause the
entire solution in the Erlenmeyer flask to change from colorless to a very pale pink.
MATERIALS AND EQUIPMENT
 50-mL burette
 5-mL volumetric pipette
 pipette bulb
 0.1 M sodium hydroxide (NaOH)
 Vinegar
 Phenolphthalein
 burette stand
 TWO 250-mL (or 125 mL)
 Erlenmeyer flasks
 wash bottle with distilled water
 funnel

PROCEDURE:

1. Obtain a 50-mL burette, 5-mL volumetric pipette and a pipette bulb from the
stockroom.

Setting up the burette and preparing the NaOH

2. Rinse the inside of the burette with distilled water. Allow the distilled water to
drain out through the tip in order to ensure that the tip is also rinsed.

3. Now rinse the burette with a small amount of NaOH. To do this, add about 5-
mL of NaOH to the burette, then twirl the burette on its side (over the sink) to
rinse its entire inner surface.
Then allow the NaOH to drain out through the tip.

4. Fill the burette with NaOH up to the top, between 0-mL and 5-mL. Use a funnel
to do this carefully, below eye-level, and preferably over the sink. After this you
will need to flush the tip of the burette. Now measure the volume at the level of
the NaOH precisely, and record it as the “Initial Burette Reading” on your report.
Also record the exact molarity of the NaOH, which is labeled on the stock bottle.

Preparing the vinegar sample

5. The volumetric pipette used in this lab is designed to measure and transfer
exactly 5.00 mL of solution. First, rinse the inside of the volumetric pipette with
distilled water. Using the pipette bulb, draw the water into the pipette up above
the 5-mL mark, then allow it to drain out through the tip. You may want to do this
several times for practice. Then perform a final rinse, but this time use vinegar.

6. Now use the volumetric pipette to transfer 5.00-mL of vinegar into a clean 250-
mL Erlenmeyer flask. Record this volume of vinegar (precise to two decimal
places) on your
report. Then add about 20-mL of distilled water and 5 drops of phenolphthalein to
this Erlenmeyer flask.

Performing the titration

7. Begin the titration by slowly adding NaOH from the burette to the vinegar in the
Erlenmeyer flask. Swirl Erlenmeyer flask as you add the base in order to
 efficiently mix the chemicals. Some pinkness may appear briefly in the flask as
the base is added, but it will quickly disappear as the flask is swirled.

8. As the equivalence point is approached, the pink color will become more
pervasive and will take longer to disappear. When this occurs, start to add the
NaOH drop by drop.
Eventually the addition of just one drop of NaOH will turn the solution in the
Erlenmeyer flask a pale pink color that does not disappear when swirled. This
indicates that the equivalence point has been reached. Do not add any more
NaOH at this point. Measure this volume of NaOH precisely, and record it as the
“Final Burette Reading” on your report.

9. Refill your burette with NaOH, and then repeat this procedure for a second
sample of vinegar, and then a third sample of vinegar. You do not need to flush
the tip of the burette again. Note that if you use less than 25-mL of NaOH for the
second titration, you do not need to refill the burette for the third titration; also that
you will need to clean out and re-use one of your Erlenmeyer flasks for the third
titration. You and your partner should take turns performing these titrations.

10. When finished, dispose of your chemical waste as instructed.

CALCULATIONS

Molarity of Acetic Acid in Vinegar

First, using the known molarity of the NaOH (aq) and the volume of NaOH (aq) required to reach the
equivalence point, calculate the moles of used in the titration.

From this mole value (of NaOH), obtain the moles of HC2H3O2 in the vinegar sample, using the
mole-to-mole ratio in the balanced equation.

Finally, calculate the molarity of acetic acid in vinegar from the moles of HC2H3O2 and the volume of
the vinegar sample used.
Mass Percent of Acetic Acid in Vinegar

First, convert the moles of HC2H3O2 in the vinegar sample (previously calculated) to a mass of
HC2H3O2, via its molar mass.

Then determine the total mass of the vinegar sample from the vinegar volume and the vinegar
density. Assume that the vinegar density is 1.000 g/mL (= to the density of water).

Finally, calculate the mass percent of acetic acid in vinegar from the mass of HC2H3O2 and the mass
of vinegar.
ACTIVITY 1
TITRATION OF VINEGAR

Fill out the table with laboratory observations and data. Answer the post-lab questions.

Trial 1 Trial 2
Initial Burette reading

Final Burette reading

Volume of NaOH used

Molarity of NaOH used

Volume of Vinegar used

Color at equivalence
point

1. Compute the molarity (3pts) and mass percent (3pts) of acetic acid in
vinegar.

2. What was the purpose of the phenolphthalein indicator in this

experiment? Be specific.

3. Suppose you added 40 mL of water to your vinegar sample instead of 20

mL. Would the titration have required more, less or the same amount of
NaOH for a complete reaction? Explain (3 pts.)
ACTIVITY 2
SPECTROPHOTOMETRY
OBJECTIVES:
To be able to use a spectrophotometer and perform a simple calibration.

INTRODUCTION:
A spectrophotometer is used to measure the light transmitted by a solution to
determine the concentration of the light-absorbing substance in the solution. The
reliability of the result may be maintained by performing calibration which is a
process of comparing measurements of known substance (standard) and unknown
substance (analyte).

Components of a Spectrophotometer

Light Source
The most common source of light for work in the visible and near-infrared region is
the incandescent tungsten or tungsten-iodide lamp.

Monochromators
Isolates individual wavelengths of light to be used in measuring the unknown
analytes.

Sample Cell
Contains the substances to be measured and also the control substance to maintain
quality results.

Photodetectors
The purpose of the detector is to convert the transmitted radiant energy into an
equivalent amount of electrical energy.

An example of single-beam spectrophotometer.

MATERIALS:

Spectrophotometer
Cuvettes
Red food coloring solution
Water

PROCEDURE:
1. Set the wavelength to 380 nm. Make sure that the sample chamber is empty
and the lid is closed. Set the display to 0% transmittance.
2. Prepare 2 cuvettes. One containing pure distilled water in millicube (blank)
and the other containing the red food coloring solution.
3. Make sure that the cuvette is clean and free from finger prints.
4. Place the cuvette blank (with distilled water) in the sample chamber.
5. Adjust the display to 100% transmittance.
6. Remove the cuvette with the blank solution.
7. Now set the display mode to absorbance by pressing the mode control key.
8. Repeat step 3 and 4 but this time using the cuvette with red food color
solution.
9. Always note for the absorbance and press the mode control key to look for
transmission. Record results.
10. Increase the wavelength by 25 nm to 405 nm.
11. Wipe the cuvette to remove dirt and finger print. (distilled water) (for another
calibration)
12. Place the cuvette in the sample chamber.
13. Adjust the transmittance control knob until the display is 100%.
14. Place the sample cuvette (red food colored solution). Record results.
15. Increase the wavelength by 25 nm to 430 nm. Repeat the steps for blank and
test substance. Record results.

DEFINITION OF TERMS:

WAVELENGTH-

ABSORBANCE-

TRANSMITTANCE-
ACTIVITY 2
SPECTROPHOTOMETRY
Fill out the table with your results and observation.

WAVELENGTH ABSORBANCE TRANSMITTANCE

QUESTIONS:

1. What is the difference between absorbance and transmittance? (4 pts)

2. Why is it important that you wipe the cuvette properly before placing it
in the sample chamber? (2pts.)

3. How does the change in wavelength affects the results? (2pts.)

4. What is the importance of using reagent blank in every measurement of

an analyte? (2pts.)
 ACTIVITY 3
Breathing and Acid-base balance

OBJECTIVES
To explain the process of acid-base balance in the body using sodium bicarbonate
solution and breathing.

INTRODUCTION

This experiment will demonstrate the role of the bicarbonate buffer system in
regulating carbon dioxide-based acidity in the body.

MATERIALS AND REAGENTS:

2 250mL beaker
100 mL graduated cylinder
10 mL graduated cylinder
Dropper
straw
Distilled water
Saturated sodium bicarbonate solution
Phenol red

PROCEDURE:

1. Use the permanent marker to label two 250 mL beakers as 1 and 2.

2. Use the 100 mL graduated cylinder to measure and pour 50 mL of distilled

water into Beaker 1.

3. Use the 100 mL graduated cylinder to measure and pour 48 mL of distilled

water into Beaker 2.

4. Use the 10 mL gradated cylinder to measure and pour 1 mL of saturated

sodium bicarbonate solution into Beaker 1. Then, add 2 drops of phenol red to
Beaker 1. Gently mix the solution.

5. Use the straw to blow bubbles into Beaker 1. Start the stopwatch as soon as
you start blowing into the straw.
 Observe the color of the solution as you blow into the straw, and record how
long it takes for the pH indicator (phenol red) to change from pink/red (basic) to
yellow (acidic).Take care not to become lightheaded as you blow into the straw. If you
become light headed, stop breathing into the straw. Continue only when you no longer feel
lightheaded. Blow into the straw for no more than 5 minutes. If you do not see a change
within 5 minutes, record "no change" as your observation.

 Record your observations in Table 1.

 Use the 10 mL graduated cylinder to measure and pour 1 mL of saturated

sodium bicarbonate solution into Beaker 2.

 Use a pipette to add five drops of the sodium bicarbonate buffer solution to
Beaker 2. Then, add 2 drops of phenol red to Beaker 2.

 Use the straw to blow bubbles into Beaker 2. Start the stopwatch as soon as
you start blowing into the straw.
ACTIVITY 3
Breathing and Acid-base balance

Fill out the table with the results and observation. Answer the questions
below.

Beaker 1 Beaker 2
Color changes

Times required for

changes to occur

Other observations

QUESTIONS:
1. Was the time required to change the solution color different for the two
beakers? Why or why not?

2. How might increasing the amount of sodium bicarbonate buffer affect

the results of the experiment?

3. What is the principle of the color change in the solution?

4. What is the one responsible for the color change?

5. How can you relate this to the acid-base balance in human body?
ACTIVITY 4
Lipid calculations
OBJECTIVES

To classify lipids and fatty acids commonly found in household and apply qualitative
analysis of lipids and fatty acids.

MATERIALS AND REAGENTS:

14 Clear test tubes

Test tube rack
Spatula
dropper
Methylene chloride
1 % Bromine solution
deionized water
Olive oil
safflower oil
stearic acid
oleic acid
lecithin
cholesterol
vitamin A.

PROCEDURE

Part 1 - Physical Properties of Lipids and Fatty Acids

1. Label 7 clean, dry test tubes. Put a small sample of each of the following
lipids in
separate test tubes: olive oil, safflower oil, stearic acid, oleic acid, lecithin,
cholesterol and vitamin A. If the lipid is a solid, use a very small amount on
the
tip of a spatula. If the lipid is a liquid, use 5 drops. If the vitamin A is given as
a
capsule, you will need to puncture it and squeeze out some of the liquid
inside to
test it.
2. Classify each of the lipids as a triglyceride, fatty acid, steroid, or
phospholipid.
3. Describe the appearance and odor, if any, of each of the lipids.
4. Add 20 drops of methylene chloride to each tube and shake each of the
tubes to
mix the solutions well. Determine whether each of the lipids is soluble or
insoluble in methylene chloride. Record your observations on the report
sheet. Save these solutions for part 2.
5. Label 7 clean test tubes. (The tubes do not have to be dry this time.) Put a
small
sample of each of the lipids in separate test tubes, as you did in step 1.
 6. Add about 2 mL of deionized water to each test tube and shake each tube to
mix
thoroughly. Determine whether each of the lipids is soluble or insoluble in
water.
Record your observations on the report sheet.

Part 2 - Bromine Test

1. For this part, you will need the samples from step 4. To each solution, add
1%
bromine solution drop by drop, shaking the tube after each drop, until the
solution
remains orange. If you add 1 drop of bromine and the orange color of
the bromine does not disappear, do not add any more bromine.

If your sample contains no double bonds, it will not react with

bromine and the orange color of bromine will not disappear, even if
only one drop of bromine is added. If your sample contains double
bonds, it will react with the added bromine, and the orange color will
disappear. The more double bonds present in the sample, the more
bromine will be needed to react with the sample. The orange color of
bromine remains in the solution when all of the double bonds have
reacted.

2. Record your observations – you will need to notice whether the orange
color
fades rapidly or persists in each case.
ACTIVITY 4
Lipid calculations
Fill out the table with the results and observation. Answer the questions below

Oliv Safflowe Steari Olei lecithi cholestero Vitami

e oil r oil c acid c n l nA
acid
Lipid
classificatio
n

appearance

odor

Solubility
with
methylene
chloride
Solubility
with
deionized
water
Color
change with
bromine

QUESTIONS:
1. What functional group is present in a triglyceride?

2. What functional group is present in a fatty acid?

3. Draw the structure of oleic acid.

4. Draw the structure of glyceryl triolein.

5. What do lipids have in common?

6. What type of solvent would be needed to remove an oil spot? Why?

7. The melting point of stearic acid is 70°C, and the melting point of oleic
acid is 4°C. Explain in detail why their melting points are so different.

8. Based on part 2 of the experiment, which oil is more unsaturated,

safflower oil or olive oil? Explain.

9. Which should have a higher melting point, safflower oil or olive oil?
Explain your
reasoning.

10. What components are present in a phosphoglyceride?

ACTIVITY 4
Lipid calculations
OBJECTIVES

The aim of this experiment is to calculate the saponification number of the oil sample
and the average molecular weight of triacylglycerols.

INTRODUCTION

The simplest lipids constructed from fatty acids are the triacylglycerols, also
referred to as triglycerides, fats, or neutral fats. Triacylglycerols are composed of
three fatty acids each in ester linkage with a single glycerol. Triacylglycerols are
major components of the natural lipid in biological tissue. Depending on the nature of
the constituent fatty acids, they may be either oils or low melting point solids. Plant
triacylglycerols generally tend to be oils, reflecting a relatively high content of
saturated fatty acids, whereas animal triacylglycerols are usually solid. Information
about the structure of triacylglycerols can be obtained by two parameters:
- Saponification Number
- Iodine Number

The saponification number gives an indication of the average molecular weight of the
triacylglycerols, while the iodine number gives a relative measure of the degree of
unsaturation. Analysis of acylglycerols can be achieved by hydrolysis followed by
isolation and characterization of products. Hydrolysis of acylglycerols is most easily
accomplished in hot alkali (saponification) but as lipids.

MATERIALS

Round bottom flask (RBF)

Distilled water
Weighing scale
Oven
Graduated cylinder
Condenser (reflux apparatus = RBF + condenser)
Erlenmeyer flask
dropper

Acetone
Oil
Boiling chips
0.5 M ethanolic NaOH
Phenolphthalein
0.5M HCl
PROCEDURE

1. Get a round bottom flask (RBF). Clean your RBF, rinse it with distilled water,
then with acetone and allow to dry in the oven.

2. After RBF cool down, weigh it and record the weight value.

3. Measure 2 mL of oil with a graduated cylinder.

4. Place the oil into to RBF, weigh your RBF once again and record the
difference as the weight of your oil sample.

5. Set up a reflux apparatus (RBF+condenser). DO NOT forget to add boiling

chips.

6. Add 20 mL of 0.5M ethanolic NaOH into the RBF.

7. Reflux for 30 min. DO NOT forget to flow water through the condenser.

8. After reflux, transfer your solution into an erlenmeyer flask and add 1-2 drops
of phenolphthalein as an indicator.

9. Titrate the solution with 0.5M HCl.

10. Record your end-point.

ACTIVITY 4
Lipid calculations
Fill out the table with the results and observation. Answer the questions below

RBF weight value

Weight of RBF with oil

Reaction after adding phenolphthalein

End-point result

QUESTIONS

1. Write the reaction involved during the reflux process.

2. Define the saponification number.

3. Calculate the saponification number of your oil sample.

4. Calculate the average molecular weight of triglycerides of your oil sample.

5. Compare your saponification number with known values of different types of

oils.
 ACTIVITY 5
ROUTINE URINALYSIS
OBJECTIVES

To be able to perform routine urinalysis.

INTRODUCTION
The basic (routine) urinalysis consists of four parts: specimen evaluation,
gross/physical examination, chemical screening, and sediment examination.
Specimen evaluation ensures that the specimen is viable and without contamination.
In physical testing, color, transparency, and specific gravity is noted. Chemical
screening includes the presence of pH, sugar, albumin and etc. The sediment testing
involves microscopic examination. We are noting the presence of pus cells, rbc,
bacteria, epithelial cells and etc.

MATERIALS

1. Centrifuge
2. Test tubes
3. Transfer pipets
4. Urine strips
5. Slides
6. Cover slips

PROCEDURE

1. Pour 10–15 mL of a well-mixed urine specimen into a graduated disposable

centrifuge tube. Perform physical examination and reagent strip chemical evaluations.
Centrifuge at 450 g for 5 min
2. Carefully remove and save the supernatant. The final volume used to resuspend the
sediment may vary with the standardized system used but should remain constant
within any given laboratory. Use a disposable pipet, specialized tube, or pipet system to
concentrate the sediment.
3. Gently resuspend the sediment in the remaining supernatant, and add one drop of
supravital stain if desired. Using an appropriate pipet, load/charge the examination
chamber of a standardized slide. Allow the urine to settle for 30–60 seconds.
4. Examine with low- and high-power objectives. Subdued light or phase-contrast
illumination will be required to detect sediment entities with a low refractive index. The
fie focus should be varied continuously while scanning. Systematically progress around
the entire examination chamber, being careful to examine along the edges for casts.
5. Count the number of casts in at least 10 lpf, average, and report the number of casts
per lpf. A reasonable range may be used in reporting (e.g., 0–2, 2–5, 5–10). Use high
power to identify casts by type. Casts will not be missed if phase-contrast microscopy is
used
6. Identify and count erythrocytes, leukocytes, and renal epithelial cells using the high-
power objective. Count at least 10 hpf, average, and report as cells/hpf. A reasonable
range may be used for reporting.
7. Comment on the following:
a. Squamous and transitional cells if present in large numbers or as fragments
(transitional cells).
b. Bacteria, yeast, and microorganisms. Bacteriuria detectable on low power should be
reported as at least 2 +.
c. Crystals (quantitated under low power). The presence of abnormal crystals should be
confimed chemically and correlated with the patient history.
d. Large amounts of mucus.

8. The authors recommend confiming the following results with cytopathologic

examination or specific chemical tests (crystals):
a. More than two renal epithelial cells/hpf.
b. Pathologic casts.
c. Atypical mononuclear cells, particularly urothelial cells.
d. Tissue fragments.
e. Pathologic crystals.
ACTIVITY 5
ROUTINE URINALYSIS
color PUS cells
transparency RBC
pH bacteria
Specific gravity Epithelial cells
sugar Mucus threads
albumin Amorphous
substances
casts
crystals
others
Fill out the table with the results and observation. Answer the questions below

QUESTIONS:
1. How does the urine color relate to your kidney function?

2. What does the presence of RBC indicate?

3. What does the presence of WBC indicate?

4. Is it normal to have bacteria in urine? Why? Why not?

5. What does the presence of casts indicate?

6.
ACTIVITY 6
Refractometer
OBJECTIVES

To measure specific gravity using refractometer and apply appropriate correction

calculation.

INTRODUCTION

Refractometer is an indirect method. The refractive index of a solution is related to

the content of dissolved solids present. The index is the ratio of the velocity of light in
air to the velocity of light in a solution. It varies directly with the proportion of particles
in solution and, therefore, with the specific gravity. The clinical refractometer is a
device that requires only a few drops of
urine (unlike the 15 mL of urine necessary with the urinometer). Although the
refractometer measures the refractive index of a solution, the scale used is valid only
for urine and cannot be used to indicate the specific gravity of salt or sugar solutions.

This should be kept in mind if salt solutions are to be used for calibration.

Special graphs or tables are required to convert refractive index scale numbers to
solute concentrations in aqueous solutions if this should be required (American
Optical Catalog Number 10403). The specific gravity reading on the refractometer is
generally slightly lower than a urinometer reading on the same urine specimen by
about 0.002.

A temperature-compensated hand model is available. The

instrument is temperature compensated between 60° and 100° F (15° to
38° C). It is damaged by heat above 150° F (66° C) and by immersion of
the eyepiece and focusing ring in water. It should read zero with distilled
water; the zero reading can be reset if necessary by breaking the seal over
the setscrew, turning it with a small screwdriver, and resealing. Check
calibration daily. Copper sulfate solutions can be adjusted to monitor a high
specific gravity level as an additional check.

Temperature corrections:
Cold specimen
Subtract 0.001 for every 3ºC that the specimen temperature is BELOW urinometer
calibrated temperature

Warm specimen
Add 0.001 for every 3ºC that the specimen temperature is ABOVE urinometer
calibrated temperature

For glucose and protein

1 g/dL glucose (-) 0.004
1 g/dL protein (-) 0.003

MATERIALS:

Refractometer
Distilled water
Cold distilled water
Hot distilled water
Sugar
Specimen

PROCEDURE
1. To make a specifcic gravity determination of urine, clean the surfaces of
the cover and prism with a drop of distilled water and a damp cloth, and
allow them to dry.
2. Close the cover. Hold horizontally, and apply a drop of urine at the notched
bottom of the cover, so that it flows over the prism surface by capillary action.
3. Point the instrument toward a light source at an angle that gives optimal
contrast.
4. Rotate the eyepiece until the scale is in focus. Read directly on the specific
gravity scale the sharp dividing line between light and dark contrast.
5. The entire procedure should be repeated with a second drop of urine from the
same sample.

ACTIVITY 6
 Refractometer
Fill the table with initial reading from the experiment. Calculate the corrected
value for specific gravity.
Distille Urine Urine Cold Hot Sugar
d water trial 1 trial 1 water water dissolved in
water

Correcte
d specific
gravity

Show your solutions here.

 ACTIVITY 7
Rule of three and Red Blood cell indices

OBJECTIVES
To be able to perform manual microhematocrit.
To apply the calculation using the rules of three and the RBC indices.

INTRODUCTION
The Hematocrit

The Hematocrit reflects the concentration of red cells—not the total red cell mass. Typical
reference values of hematocrit for adult males are 0.41–0.51 and 0.36–0.45 for females. A
value below an individual’s normal value or below the reference interval for age and sex
indicates anemia, and a higher value, polycythemia. The Hematocrit is low in hydremia of
pregnancy, but the total number of circulating red cells is not reduced. The Hct may be
normal or even high in shock accompanied by hemoconcentration, although the total red cell
mass may be decreased considerably owing to blood loss. The Hct is unreliable as an
estimate of anemia immediately after loss of blood or immediately following transfusions.
The rule of three
3 x RBC = Hb 3 x Hb = HCT ± 3 (%)
Erythrocyte indices

Wintrobe introduced calculations for determining the size, content, and Hb concentration of
red cells; these erythrocyte indices have been useful in the morphologic characterization of
anemias. They may be calculated from the red cell count, Hb concentration, and Hct.

Mean Cell Volume

MCV - the average volume of red cells

- calculated from the Hct and the red cell count.
- expressed in femtoliters or cubic micrometers.

MCV Hct X1000/RBC (in millions per L)

Example:

Hct = 0.45
red cell count = 5 10^12/L
Mean Cell Hemoglobin

MCH - content (weight) of Hb of the average red cell

- it is calculated from the Hb concentration and the red cell count.
- value is expressed in picograms.

MCH = Hb (in g /L) / RBC (in millions /µL)

Example:

Hb = 150 g/dL
Red cell count = 5 × 10^12/L

Mean Cell Hemoglobin Concentration

MCHC - is the average concentration of Hb in a given volume of packed red cells.

-calculated from the Hb concentration and the Hct.

The 95% reference intervals for normal adults are as follows:

MCV = 80–96 fL
MCH = 27–33 pg
MCHC = 33–36 g/dL

In microcytic anemias, the indices may be as low as an MCV of 50 fL, an MCH of 15 pg, and
an MCHC of 22 g/dL; rarely do any become lower.

In macrocytic anemias, the values may be as high as an MCV of 150 fL and an MCH of 50
pg, but the MCHC is normal or decreased .The MCHC typically increases only in
spherocytosis, and rarely is over 38 g/dL.

MATERIALS
heparinized capillary tube
Microhematocrit centrifuge
Millimeter rule
PROCEDURE
Hematocrit Measurement by Micromethod

1. The microhematocrit tube is filed by capillary attraction from a free flowing puncture
wound or a well-mixed venous sample.
2.The capillary tube should be filled to at least 5 cm.
3. The empty end is sealed with modeling clay.
4. The filled tube is placed in the radial grooves of the microhematocrit centrifuge head with
the sealed end away from the center.
5. Place the bottom of the tube against the rubber gasket to prevent breakage.
6. Centrifugation for 5 minutes at 10,000–12,000 g is satisfactory unless the Hct exceeds
50%;
in that case, an additional 5 minutes’ centrifugation should be employed to ensure minimal
plasma trapping. The capillary tubes are not graduated.
7.The length of the blood column, including the plasma, and of the red cell column alone
must be measured in each case with a millimeter rule and a magnifying lens, or with one of
several commercially available measuring devices. The instructions of the manufacturer
must be followed.
 ACTIVITY 7
Rule of three and Red Blood cell indices

Actual Hct Hemoglobin RBC Mean Cell Mean Cell Mean Cell
measurement Volume Hemoglobin Hemoglobin
Concentration

Fill the table with the results obtained from activity. Answer the questions that follow.
Show your solutions here:

QUESTIONS:
1. What causes the increase in hematocrit values?

2. Explain the relationship between hemoglobin.

3. What is the terminology used when MCV is at 80-100 fL?, <80 fL?, >100 fL?

4. What is the terminology used when MCHC is at 30-37 g/dL?, <30 g/dL?, >37
g/dL?

5. In what conditions do you usually encounter high MCV? Low MCV?

ACTIVITY 8
Manual wbc count and NRBC corrections

OBJECTIVES

To be able to perform manual WBC count and apply correction when there’s
appearance of NRBC.

INTRODUCTION

Specimen Collection
EDTA should be used; heparin is unsatisfactory as an anticoagulant.

Hemocytometer Method
Although this method is used only occasionally in leukocyte counting, the
technologist should be able to perform it

Counting Chamber. The hemocytometer is a thick glass slide with

inscribed platforms of known area and precisely controlled depth under
the coverslip. Counting chambers and cover glasses should be rinsed in
lukewarm water immediately after use; wiped with a clean, lint-free cloth;
and allowed to air dry. The surfaces must not be touched with gauze or
linen because these materials may scratch the ruled areas.

Diluting Fluid. The diluting fluid lyses the erythrocytes so that they will
not obscure the leukocytes. The fluid must be refrigerated and filtered
frequently to remove yeasts and molds

Importance of knowing WBC manual count:

1. As a check on the validity of electronic methods for calibration
purposes
2. As a check on the validity of electronic counts in patients with
profound leukopenia or thrombocytopenia
3. For blood specimens with platelet counting interference (i.e., very
microcytic RBCs), and
4. As a backup method.
It is also commonly used as a method for counting cells in cerebrospinal
fluid (CSF).

Nucleated Red Cells

Nucleated red cells are precursors of non-nucleated mature red cells in the blood. In
the human, normoblasts are normally present only in the bone marrow. Stages in
their production from the earliest to the latest include pronormoblast, basophilic
normoblast, polychromatophilic normoblast, and orthochromatic normoblast. In
general, nucleated red cells that might appear in the blood in disease are
polychromatic normoblasts. In some, however, the cytoplasm is so basophilic that it
is diffiult to recognize the cell as erythroid except by the character of the nucleus,
intensely staining chromatin, and sharp
separation of chromatin from parachromatin. The megaloblast is a distinct, nucleated
erythroid cell—not merely a larger normoblast. It is characterized by large size and
an abnormal “open” nuclear chromatin pattern.

MATERIALS

venous blood (EDTA)

Hemocytometer
Microscope
Ticker
Cover glass
WBC Diluting fluid ( 2-3 % glacial acetic acid or 1% HCl or tuerk’s solution)
WBC Thoma pipette (with syringe barrel)
PROCEDURE
Leukocyte Counts: Manual

1. Suck blood to .5 mark of pipette.

2.Suck diluting fluid to 11 mark of thoma pipette.
3.Shake pipette to mix.
4.Discard first few drops.
5.Charge counting chamber.
6. Count WBC in 4 large square corners.
7. Compute cells as follows:

WBC counted x 10 x 20
WBC in thousand/mm
3
= 4

Where: 10 = depth correction factor (constant)

20 = dilution factor (variable)
4 = area correction factor (constant)

Volume∈thebulb (c onstant 10 for WBC )

*Dilution Factor =
volume of blood used

Nucleated Red Blood Cells.

Nucleated red blood cells (NRBCs) will be counted and cannot be distinguished from
leukocytes with the magnification used. If their number is high, as seen on the
stained smear, a correction should be made according to the following formula:

where the No. of NRBCs is the number of nucleated red cells that are counted during
the enumeration of 100 leukocytes in the differential count.
ACTIVITY 8
Manual wbc count and NRBC corrections

Fill the table with results and observations.

Initial WBC count

Calculated WBC count

Corrected WBC count (due to NRBC)

Show your computation:

QUESTIONS:
1. What is the importance of the diluting fluid?

2. Why do you need to discard the first few drops before charging the mixture
to the hemocytometer?

3. Is it abnormal to have a presence of NRBC in bone marrow? Why? Or Why

not?

4. What is the normal range of NRBC?

5. In what condition do you usually see the presence of NRBC in peripheral

blood smear?

ACTIVITY 9
Reticulocyte count
OBJECTIVES

To be able to perform reticulocyte counting properly and accurately.

INTRODUCTION

Principle
Reticulocytes are immature non-nucleated red cells that contain ribonucleic acid (RNA) and
continue to synthesize Hb after loss of the nucleus. When blood is briefly incubated in a
solution of new methylene blue or brilliant cresyl blue, the RNA is precipitated as a dye–
ribonucleoprotein complex.

Microscopically, the complex appears as a dark blue network (reticulum or fiamentous

strand) or at least two dark blue granules that allow reticulocytes to be identified and
enumerated.

Reference Values
Normal adults have a reticulocyte count of 0.5%–1.5%, or 24–84 × 109/L. In newborn
infants, the percentage is 2.5%–6.5%; this falls to the adult range by the end of the second
week of life.

Interpretation
Because reticulocytes are immature red cells that lose their RNA a day or so after reaching
the blood from the marrow, a reticulocyte count provides an estimate of the rate of red cell
production. An absolute reticulocyte count or reticulocyte production index is more helpful
than the percentage.

MATERIALS

 1% new methylene blue in a diluent of citrate/saline (or brilliant cresyl

blue)
(one part 30 g/L sodium citrate plus four parts 9 g/L sodium chloride).
 Oil immersion
 Test tube
 Slide
 Microscope

PROCEDURE

1. Three drops each of reagent and blood are mixed in a test tube
2. Incubated for 15 minutes at room temperature, and remixed. Two wedge fims are
made on glass slides and air dried.
3. Viewed microscopically with an oil immersion lens, reticulocytes are pale blue
and contain dark blue reticular or granular material, and red cells stain pale blue
or blue-green.
4. The percentage of reticulocytes is determined in at least 1000 red cells.

COMPUTATION:

No. of retics counted

% retics = No . of RBC counted ( 1000 ) x 100

Example:
No. of retics counted: 20
No. of RBC counted: 1000

20
% retics = 1000
x 100 = 2.0 %
ACTIVITY 9
Reticulocyte count
Write the number of reticulocyte counted and calculate % retics. Show your
computation.

QUESTIONS:
1. What is the principle of reticulocyte counting?

2. What is the importance of using new methylene blue or brilliant cresyl

blue?

3. Describe the appearance of reticulocyte.

4. What does having elevated and decreased amounts of reticulocyte

indicate?

5. In what condition do you usually encounter elevated and decreased

amounts of reticulocyte?
 ACTIVITY 10
Colony counting and antimicrobial
susceptibility testing
OBJECTIVE

To be able to perform bacterial planting, calculate colony forming units and execute
antibacterial susceptibility testing.

INTRODUCTION
Antimicrobial susceptibility testing is performed on bacteria isolated from clinical
specimens to determine which antimicrobial agents might be effective in treating infections
caused by the bacteria. Only bacteria that are likely to be contributing to an infection should
be tested. Testing bacteria that are not involved in the infection would be misleading to the
physician and could lead to a more serious infection with development of antimicrobial
resistance. One of the major challenges in clinical microbiology is the identification of the
bacterium or bacteria that are the cause of infections. Often, these bacteria need to be
distinguished from the flora that may reside at the site of the infection normally, although in
some situations the microbial flora that reside at the site of the infection may be contributing
to the infection. Therefore, thought needs to go into determining which bacterium or bacteria
from a specimen will be tested for susceptibility to antimicrobials.

In clinical laboratories, susceptibility testing is usually performed by a disk diffusion or

dilution (minimal inhibitory concentration [MIC]) method. Standards that describe these
methods are published and frequently updated by the Clinical and Laboratory Standards
Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards
[NCCLS]).
Clinical laboratories can perform testing according to recommendations in the CLSI
standards or use one of several different types of commercial manual or automated
antimicrobial susceptibility test systems. On a worldwide level, antimicrobial susceptibility
testing is guided by the International Standards Organization (ISO).

Example of reporting protocol in susceptibility

Testing. We use the word “susceptible” and
“resistant”.

MATERIALS:

1. Petriplate containing microbial culture(For example, Escherichia coli)

2.  Inoculation loop
3. Bunsen burner
4. Saline solution
5. McFarland solution
6. MHA plate
7. Cotton swab
8. Antibiotic disks(Streptomycin (S), Ciprofloxacin (CIP), Chloramphenicol (C),
Doxycycline (D), Penicillin G (P), Gentamycin (G)
9. Tooth pick
10. Incubator
11. Vernier caliper
12. Sterile test tube

PROCEDURE
 
1. Select a pure culture plate of one of the organisms to be tested.
2. Aseptically emulsify a colony from the plate in the sterile saline solution. Mix it
thoroughly to ensure that no solid material from the colony is visible in the saline solution.
3. Repeat until the turbidity of the saline solution visually match that of the standard
turbidity.
4. Take a sterile swab and dip it into the broth culture of organism.
5. Gently squeeze the swab against the inside of the tube in order to remove excess
fluid in the swab.
6. Take a sterile Mueller-Hinton agar (MHA) plate or a nutrient agar (NA) plate.
7. Use the swab with the test organism to streak a MHA plate or a NA plate for a lawn
of growth.
8. After the streaking is complete, allow the plate to dry for 5 minutes.
9. Antibiotic discs can be placed on the surface of the agar using sterilized forceps.
10. Gently press the discs onto the surface of the agar using flame sterilized forceps or
inoculation loop.
11. Carefully invert the inoculated plates and incubate for 24 hours at 37° C.
12. After incubation, use a metric ruler to measure the diameter of the zone of inhibition
for each antibiotic used.
13. Compare the measurement obtained from the individual antibiotics with the standard
table to determine the sensitivity zone.

ACTIVITY 10
 Colony counting and antimicrobial
susceptibility testing

Fill the table with the results and observation. Answer the questions that follow.
Number Total Streptom Ciprofloxa Chloramphe Doxycyclin Penicillin Gentamycin
of pure no. of ycin (S) cin (CIP) nicol (C) e (D) (P) (G)
colony colony (Zone of (Zone of (Zone of (Zone of (Zone of (Zone of
forming inhibition) inhibition) inhibition) inhibition) inhibition) inhibition)
unit

QUESTIONS:
1. Why is it important to make sure that you are counting the pure colony and not
the contamination?

2. What is the importance of McFarland standard?

3. Can you use other medium that MH agar? Why? Why not?

4. Why is it that you should wait for about 5 mins. to dry the streak before putting
the antibiotic discs?

5. Compare the inhibitions of the antibiotic discs.