Shifting Exam Finals Trans

analytical variable including analytical (equipment and
reagent problems) or technical errors during analysis to

CLINICAL MICROSCOPY prevent reporting of incorrect individual results
• By performing and adhering to QC procedures, a
CLINICAL MICROSCOPY laboratory provides a high quality services by removing
the needs of laboratory users
• Performs scientific analysis of non-blood body fluids
Specimen Handling
• The macroscopic, chemical, and microscopic
examinations of urine provide initial valuable diagnostic
• Preserve the specimen by refrigeration or use of an
information concerning metabolic dysfunction of both
appropriate chemical preservatives
renal and non-renal origin
• If not properly preserved, the following changes may
• Helps clinician in early detection of gastrointestinal
occur:
bleeding, liver, and biliary duct disorders and
o Increased analyte:
malabsorption syndrome
1. pH
Tests offered: 2. bacteria
3. odor
1. Routine urinalysis 4. nitrite
2. Routine fecalysis o Decreased analyte:
3. Pregnancy test 1. clarity
4. Fecal occult blood test 2. glucose
5. Micral test 3. ketones
6. Sperm analysis 4. bilirubin
5. urobilinogen
Standard Precaution 6. RBCs and WBCs
• Gloves must be worn at all times HISTORY AND IMPORTANCE

• Specimen must be collected in clean, dry, leak-proof
containers • Analyzing of urine was actually the beginning of
• Properly applied screw-top lids are less likely to leak than laboratory medicine
snap-on lids • References to the study of urine can be found in the
• Containers must be: drawing of caveman and in Egyptian hieroglyphics, such
o Wide mouth with wide, flat bottom to prevent as the Ewin Smith surgical papyrus
overturning • Pictures of early physicians commonly showed them a
o Made of clear materials go allow for bladder-shaped flask of urine
determination of color and clarity • Although these physicians lacked the sophisticated
o Recommended capacity is 50mL (allows 12mL testing mechanisms, they were able to obtain diagnostic
of specimen needed) information from such basic observation as color,
• All specimens must be labeled properly turbidity, odor, volume, viscosity, and even sweetness
o Labels must be attached to the containers and • Fifth century BC: Hippocrates wrote a book on
not to the lid “Uroscopy”
• Specimen rejection: • 1140 AD: Color charts had been developed that
o Specimen in unlabeled containers described the significance of 20 different colors
o Non-matching labels and requisition form • Frederik Dekkers (1694): Chemical testing progressed
o Specimens contaminated with feces or toiler from “Ant testing” and “Tase testing” for glucose and
paper discovery of albuminuria in boiling urine
o Specimen of insufficient quantity • Thomas Addis (17th century): Invention of microscope
o Specimens that have been improperly lead to the examination of urinary sediment and to the
transported development methods for quantitating microscopic
• Requisition / Request forms must accompany specimens sediments
• Specimen must be sent to the laboratory and tested • Richard Bright (1897): Introduced the concept of
within 2 hours urinalysis as part of a doctor’s routine patient examination
• 1930s: Urinalysis began to disappear from routine
Quality Control examination
• Fortunately, development of modern testing techniques
• Quality control (QC) is a system for monitoring analytical
rescued routine urinalysis, which has remained an
testing to ensure the reliability and accuracy of each
integral part of the patient’s examination
measurement performed on specimen
• Two unique characteristics of a urine specimen
• Laboratory QC procedures are designed and
account for this continued popularity:
implemented to detect pre analytical variables and
Page 1 of 38
Transcribed by: Clarence Shayne B. Nobelo
o Urine is a readily available and easily collected Food: beets, blackberries, and rhubarb
specimen Medication: Rifampin for TB (Rifadin,
o Contains information about many of the body’s Rimactane), phenazopyridine (Pyridium)
major metabolic functions laxative
Orange Hyperbilirubinemia: yellow foam when
shaken and positive chemical test
WHAT IS URINALYSIS?
Medical condition: bile duct or liver
disease and dehydration
• Commonly ordered panel of tests on a urine sample Medication: Acriflavin, phenazopyridine
• Disease associated: (Pyridium), nitrofurantoin, and
o Kidney failure phenindione
o UTI Brown Melanin / homogentisic acid: additional
Black test is recommended
o Kidney and urethral stones
Medical condition: some liver and
o GU malignancies kidney disorders and some urinary tract
o Acid-base disorders infections
o Volume status Medication: Chloroquine and
o Rhabdomyolysis primaquine (antimalarial drugs),
o Response to alkalinization therapy metronidazole (flagyl), and nitrofurantoin,
and methocarbamol (muscle relaxant)
3 Categories of Urinalysis Green Dye: used for some tests of kidney and
bladder function
1. Gross inspection / Physical test Medical condition: urinary tract
infections caused by Pseudomonas
a. Color bacteria
b. Characteristics / turbidity Medication: Amitriptyline, indomethacin
2. Dipstick / Chemical test (Indocin, Tivorbex), and propofol
a. Blood (Diprivan)
b. Bilirubin Purple Purple urine bag syndrome: research
c. Urobilinogen so far does not show it is related to the
d. Ketones type or brand of urine bags or catheters
used
e. Protein Food: others believe it is caused by a
f. Nitrite change in where tryptophan, an amino
g. Glucose acid found in many food, is broken down
h. Specific gravity in the body
i. Leukocyte estrerase Medical condition: similar to increase
j. pH spasms, autonomic dysreflexia, mild
lower back pain or other aches, fatigue,
3. Microscopy / Microscopic test
fever or chills, urinary leakage or having
a. WBCs catheterize more often nausea,
b. RBCs headache, blood or sediment in the urine,
c. Epithelial cells cloudy urine or a foul odor to the urine
d. Mucus thread White Hypercalciuria and chyluria: research
e. Bacteria so far does not show it is related to the
f. Urates type or brand of urine bags or catheters
used
g. Crystals
Others: typically caused by your body
h. Yeasts sending an increase in WBCs to fight an
i. Renal cells infection
j. Casts Medical condition: Filariasis,
Schistosomiasis, post-surgery,
GROSS INSPECTION / PHYSICAL TEST malignancy, hyperuricosuria,
phosphaturia, hyperoxaluria, proteinuria,
Color pyuria, lipiduria, caseous material from
renal tuberculosis, congenital
malformations of the lymphatic vessels
• Varies from time to time
• Very light yellow: consuming plenty of liquids
• Very dark yellow / amber: dehydrated Turbidity
• Rough guide on dehydration
• Influenced by medical condition, medication, and even • How clear or cloudy the urine is
some food • Reported in qualitative terms
• Turbid: may indicate presence of UTI or precipitated
Color Causes crystals
Red Bleeding: can be kidneys, ureters, • Never assume the presence of or absence of UTI based
bladder and urethra (source)
on turbidity
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Characteristics • Enter key: for starting or selecting a mode at each step
Clear No visible particulates, transparent • Escape key: to terminate testing from each step
Hazy Few particles, print easily seen through • Navigation key: for moving or selecting menu
urine
• Number keys: for selection of menu
Cloudy Many particulates, print blurred through the
urine
Turbid Print cannot be seen through urine Calibration:
Milky Many precipitate or be clotted
1. Enter into “System set up” by pressing “Esc” key twice.
2. Select “System configuration” and press “Ent” key.
DIPSTICK / CHEMICAL TEST 3. Navigate to “White calibration” and select “YES” press
“Ent” key.
• Consists of a series of pads embedded on a reagent strip 4. Press “Esc” key to go back to Menu.
• May vary between different manufacturers 5. Put the calibration strip on the strip load plate.
6. Result is printed through thermal printer.
How does it work? 7. Normal calibration values are between 333 ± 10 (323 –
343).
1. Take the reagent strip and dip directly to the urine
2. Wipe off excess fluid and wait a predetermined length of How to carry out the test?
time
3. Compare the colors of the individual pads against the 1. Put the test strip onto the strip loading plate after dipping
standards that are provided in urine.
2. 100 seconds later, result of first strip appears and next
Machine used: strip every 5 seconds.
• SD Urometer 720 Urine Analyzer Parameters
1. Specific gravity
• Measure of relative density
𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑢𝑟𝑖𝑛𝑒
• 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑔𝑟𝑎𝑣𝑖𝑡𝑦 =
𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟
• May vary from 1.000 to 1.030
• pKA (acid dissociation constant) charge of
polyelectrolytes
• Reading time: 45 seconds
• Clinical significance:
o Water-loss dehydration
▪ When people do not drink enough fluid
o Evaluates the body’s water balance (hydration)
Features:
o Helps evaluate kidney functions and possible
• Autocalibration with power-on kidney diseases
• Maximum 720 tests / hour o University of East Anglia (UEA): not the only test
• Easy input multiple ID and use data by keyboard used to elderly
• 2,000 tests results memory
• 10 parameters testing available
Components and keyboard:
2. pH (concentration of hydrogen ions)

• The kidneys can produce urine with a pH ranging from 5.0
to 8.0
• Double indicator system
o Diagnosing renal tubular acidosis (RTA)
o Monitoring urine alkalinization
o Aids in the elimination of certain drugs
o Differentiation of different types of kidney stones
• pH 5.0 or lower
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o Acidemia: hypoventilation, excessive • Blood in urine: hematuria
production of lactate or ketones • Pseudoperoxidase activity of hemoglobin
• pH 7.0 and 8.0 • Reading time: 60 seconds
o Alkalemia: distal RTA, UTIs secondary to • Clinical significance:
urease producing organisms (e.g., Proteus and o UTI
Klebsiella) o Kidney infection
• Regulating diet mainly controls urinary pH o Medication
• Diets rich in animal proteins: acidic urine o Strenuous exercise
• Diets composed of vegetables: alkaline urine • Early stages of bladder cancer cause bleeding – but little
• Crystals form while the urine is being produced in the to no pain, or other symptoms
kidneys: kidney stone • Infection, benign (non-cancerous) tumors, kidney or
• pH levels varies during the day, going from more acid in bladder stones, or other benign kidney diseases
the morning, to more alkaline in the afternoon / evening • False positive: menstrual contamination
3. Glucose
• Freely filtered through the glomerulus and reabsorbed in
the proximal tubule
• Glycosuria: glucose in urine
• Measured in mg/dL
• Double sequential enzymatic reaction 5. Protein
• Reading time: 30 seconds • Detection of protein in urine on urine dipstick is most
• Clinical significance: sensitive for albumin
o Hyperglycemia • Sensitivity is highly dependent upon urine concentration
o Proximal tubular dysfunction (Fanconi • Proteinuria is reported on a trace to 4+ scale
syndrome) • Protein error of indicator
o Glomerular disease: diabetic nephropathy
o Overflow proteinuria:
▪ Multiple myeloma
• Benedict’s test: confirmatory test ▪ Rhabdomyolysis
▪ Intravascular hemolysis
How is the test done?
a. Place 5mL of Benedict’s reagent into a test tube.

b. Add 5 drops (0.25mL) urine, then mix.
c. With a tong, place the test tube in boiling water bath for 5
minutes or boil over flame for 1 to 2 minutes.
• Exton’s test / Sulfosalicyclic acid test: confirmatory
d. Observe for the color change.
test
How is the test done?
a. Pipette 2mL of Exton’s reagent and place on the tube.

b. Place 4 drops of urine in the tube with Exton’s reagent.
c. Observe for cloudiness.
d. Grade the degree of turbidity using this chart.
Trace Very slight cloudiness is observed

1+ Distinct turbidity, no granulation
2+ Turbidity with granulation, no flocculation
3+ Turbidity with granulation and flocculation
e. Document your result.
4+ Clumps of protein
4. Blood
• Highly sensitive to hemoglobin 6. Leukocyte Esterase and Nitrite
• Measured in RBC/uL • Aids in the diagnosis of UTIs
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• LE is an enzyme released by WBCs • Clinical significance:
• Not directly measurement of WBC concentration, just the o Liver diseases
enzyme activity ▪ Cirrhosis
• Measured in WBC/uL ▪ Viral hepatitis
• Reading time: 2 minutes (120 seconds) ▪ Liver damage due to drugs or toxic
substances
▪ Increased RBC destruction (hemolytic
anemia)
Note: this test is only one measurement of your liver function
• Nitrites detects the presence of bacteria

• Enterococcus species lacks the ability to form nitrites
• Few reasons for negative nitrite test in urine:
o Insufficient bladder incubation time
o Decrease excretion of nitrates in the urine
• Bilirubin is a highly pigmented compound that is a by-
o Low urine pH
product of hemoglobin degradation
• Measured either positive or negative
• Bilirubin is measured in mg/dL
• Greiss reaction
• Diazo reaction
o Liver diseases: hepatitis
o Problems on liver functions
Note: urine-bilirubin measurement is common in urinalysis dipsticks, and

are known to yield a high rate of false positive results
o Diagnosis of UTIs
o Indwelling urinary catheter
7. Ketones
• β-hydroxybutyrate, acetoacetate, and acetone: act as
alternative energy carriers in the blood MICROSCOPIC EXAMINATION
• Ketoacidosis
• Measured in mg/dL Preparation of urine for microscopy:
• Sodium nitroprusside reaction
• Reading time: 40 seconds 1. Centrifuge urine for 5 minutes at speed of 1500 – 2000.
• Clinical significance: 2. Most of the supernatant is poured off and remaining pellet
o Ketoacidosis is resuspended.
▪ Poorly controlled diabetes 3. The resuspended material is called “urine sediment”.
▪ Infection and heart attack 4. Consider the storage, age, and conditions.
▪ Alcoholism and starvation
o Ketogenic diets
▪ Kids with severe epilepsy
Note: dipstick test can only detect acetoacetate and acetone

Possible findings
1. RBCs
8. Bilirubin and Urobilinogen

• Urobilinogen is the breakdown product of bilirubin and is
formed from the reduction of bilirubin
• If there is little or no urobilinogen in your urine, it can
mean your liver is not working correctly
• Measured in mg/dL
• Ehrlich reaction
• Quantified as nuclei of cells / HPF
• Lack of nuclei
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• >3 RBCs / HPF is considered abnormal
• Presence of dysmorphic RBCs strongly suggestive of 4. Mucus threads
glomerular disease
• Disease associated:
o UTIs
o Renal stones
o GU malignancies
o Coagulopathy
o Glomerular nephritis
o Sickle cell anemia
o Renal TB
o Vigorous exercise
o Contamination of menstrual blood
2. WBCs
• Thick, slimy substance that coats and moistens certain

parts of the body
• May indicate UTIs or other medical conditions
5. Bacteria
• Quantified as number of cells / HPF

• >5 / HPF is considered abnormal
• Sign of inflammation
• Disease associated:
o UTIs
o Indwelling urinary catheter
o Recent instrumentation of GU tract
o Urologic malignancy • Caused UTIs
o Chronic interstitial nephritis • Negative LE and nitrite = poor collection technique
o Interstitial cystitis
o Intra-abdominal inflammatory process adjacent 6. Crystals
to GU tract • Composed of small number of ions and molecules
o Contamination with vaginal secretions • Dependent upon the concentration of the ions and
molecules
3. Squamous epithelial cell
• Suggests that sample was contaminated

• Sample contains cells from:
o Urethra: men
o Vaginal opening: women
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7. Casts FECALYSIS / STOOL ANALYSIS
Fecalysis
• Refers to a series of laboratory tests done on fecal

samples to analyze the condition of a person’s digestive
tract in general
• Early detection of Gastrointestinal (GI) bleeding
• Liver and biliary disorders
• Long, cylindrical structures formed in the renal tubules • Maldigestion / Malabsorption syndromes
due to the precipitation of Tamm-Horsfall mucoprotein • Inflammation
• Promoted by acidic and concentrated urine • Causes of diarrhea and steatorrohea
• Identification of pathologic bacteria and parasites
Note: Mucus threads, squamous epithelial cells and bacteria = reported
from RARE, FEW, MODERATE, and MANY Specimen Collection and Handling
Processing of Routine Urinalysis • Collected in a clean container (plastic or glass) with

screw-capped tops
1. Log the incoming urine specimen in the clinical • Must not be contaminated with urine or toilet water
microscopy entry sheet and assign for accession number.
• Examine liquid specimens within 30 minutes of passage
2. Mix the urine gently by swirling and pour approximately
• Examine soft specimens within 1 hour of passage
5mL into the clean test tube.
• Examine formed stools within 24 hours
3. Observe the color and transparency or appearance of the
urine. Record it down. Physical / Macroscopic examination
4. Dip the reagent strip into the urine sample, moistening all
the pads. 1. Color
5. Remove strip into the urine immediately and tap to • Results from the intestinal oxidation of stercobilinogen to
remove the excess urine, put if possible, blot edge on the urobilin
absorbent tissue paper.
6. Put the test strip onto the strip loading plate. At the start Color Causes
time, number 5 appears on the LCD. Pale Pathologic: bile duct obstruction
7. The number decreases and when it drops to 0, the system Non-pathologic: use of Barium sulfate
will start measuring. You can put the next strip when Bloody-black Pathologic: upper GI bleeding
green light is on. tarry Non-pathologic: iron, charcoal, bismuth
Red Pathologic: lower GI bleeding
8. After 100 seconds, result of the first strip appears and
Non-pathologic: rifampin and beets
next strip every after 5 seconds. Green Seen in patients taking oral antibiotics
9. Attached the urine chemistry result in your worksheet. Ingestion of increased amounts of green
10. Discard urine sample into the biohazard container. vegetables or food coloring
11. Centrifuge urine at 1500 to 2000 rpm for 5 minutes. Use Yellow Milk diet, corn meal, rhubarb and fats
a balance tube in every centrifugation.
12. Remove tube from centrifuge after the rotor spinning.
13. Pour off supernatant urine, leaving approximately 0.5mL 2. Consistency / Appearance
of urine in tube. • Firm to soft = normal
14. Re-suspended urine sediment by tapping the bottom of • If it sways one way or another, it could suggest some
the tube. digestion or fiber issues
15. Place one drop of res-suspend urine into a clean glass
slide. Types
16. Place slide on the microscope stage and focus using low Sausage shaped with Normal
cracks in the surface
power (10x) and the condenser lowered.
Smooth, soft sausage or Normal
17. Scan 10-15 low power fields, count the number of cast (if snake
present) per field, record the average. Separate hard lumps Very constipated
18. Rotate the high-power objective (45x), into position (raise Lumpy and sausage like Slightly constipated
condenser if necessary). Scan 10-15 fields on high Soft blobs with clear-cut Lack of fibers
power. edges
19. Count the number of WBC and RBC. Mushy with ragged edges Inflammation
20. Observe the sample for the presence of microorganisms, Liquid with no solid Inflammation
pieces
crystals, mucus, epithelial cells and estimate and record
if present.
21. If crystals are present, identify type.
22. Complete the urinalysis report.
Page 7 of 38
Microscopic Examination 4. Open perforated window on the back.
5. Apply two (2) drops of Hema-Screen Developer to
• Uses a portion of stool mixed with Normal Saline Solution the back side of the boxes 1 and 2.
and Lugol’s Iodine 6. Read results after 30 seconds and within 2 minutes.
• Cover slip is placed on top of the slide and ready to be 7. Record the result; any trace of blue color within or the
viewed under the microscope outer rim of the specimen, is positive for occult blood.
• The medical technologist then looks for the following:
o Vegetable fibers MICRAL TEST
o Vegetables cells
o Fat globules • Aids in early diagnosis of microalbuminuria
o Cells such as RBC and WBC • Sensitivity: 0-10 mg/dL
o Yeast • Immunologic detention of human albumin by mean of
o Intestinal parasite soluble antibody-gold-conjugate
• Normal fecalysis result does not contain any parasites, • Urine sample is absorbed by the test strip
RBC, WBC, or any bacteria • Zone 1: conjugate fleece contains free gold-labelled
• Result of both normal and abnormal, will help the antibodies
physician in the diagnosis • Zone 2: capture matrix fleece with fixed human serum
albumin
Process of Fecalysis
1. With a pencil or marker, write the patient’s name or

number and the date at the left end of the slide.
2. Place a drop of saline in the center of the left half of the
slide and place a drop of iodine solution in the center of
the right half of the slides.
3. With the applicator stick, pick up a small portion of the
• Albumin concentration of an average urine specimen
specimen (size of a match head) and mix with the drop of
should not exceed 15 – 20 mg/dL
saline.
• A normal microalbuminuria value does not necessarily
4. In the same process with the drop of iodine.
rule out renal disease
5. Cover the drop of saline and the drop of iodine with a
cover slip. Process of Micral Test
6. Hold the cover slip at an angle, touch the edge of the
drops, and lower gently on the slide. 1. Dip the test strip into the urine for 5 seconds.
7. Record the stool color and consistency. 2. Place the strip on a nonabsorbent surface or across the
8. Put the slide with the mounts on the microscope stage top of collection cup.
and focus on the mount with the x10 or lower-power 3. Allow excess urine to drain and wait for 1 minute.
objective. 4. Compare the color of the detection pad on the strip with
9. Examine in the entire cover slip area with the x10 the color scale on the test vial.
objective. 5. Record and print your result.
10. Record and print your results.
PREGNANCY TEST
FECAL OCCULT BLOOD TEST (FOBT)
• The appearance and rapid rise in concentration of hCG in
• Refers to blood in feces that is not visibly apparent the mother’s serum and urine make it an excellent marker
• 3 chromagen indicator: for confirming pregnancy
o Benzidine: most sensitive • Can be detected as early as 8-10 days after ovulation (1
o Ortho-toluidine day after implantation) and peaks at 8-10 weeks of
o Gum guaiac: least sensitive pregnancy
• Pseudoperoxidase activity of hemoglobin • Principle: immunochromatographic assay
• Color change after 30 seconds • Sensitivity: 25 mIU/mL
Process of Fecal Occult Blood Test Process of Pregnancy Testing
1. Using the applicator sticks provided, collect a small 1. Remove the test device from the foil pouch, and place it
amount of stool specimen, on one end of the on a flat, dry surface.
applicator. Apply a very thin smear in box 1. 2. Holding the disposable dropper above the test device,
2. Reuse applicator to obtain a second sample from a squeeze 3-4 drops of serum / urine into the sample well.
different part of the stool specimen. Apply a very thin 3. As the test begin to work, you will see a purple color band
smear inside box 2. move across the result window in the center of the test
3. Allow the specimen to air dry, the close the cover. device.
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4. Interpret test result at 5 minutes. 2.1. Take note if sample is already liquefied upon receipt.
5. Record and print your result. 2.2. If the sample does not liquefy after 60 minutes,
report time as >1 hour.
SEMEN ANALYSIS 3. Determine the viscosity.
3.1. Gently aspirate the sample into a wide-bore
• The macroscopic, chemical and microscopic examination (approximately 1.5mm diameter) plastic disposable
of semen pipette allowing the semen to drop by gravity.
• Done to ascertain the total count and functional attributes 3.2. And observing the length of any thread or introduce
of semen sample a glass rod into the sample and observing the length
of the thread that forms upon the withdrawal of the
Collection and Handling: rod.
4. Measure the volume.
• Complete ejaculation should be collected in a sterile, dry, 5. Determine the pH.
wide mouth plastic container 5.1. Mix the semen sample well.
• Period of abstinence between 3-5 days is mandatory 5.2. Spread a drop of semen evenly on the pH segment
of the urine reagent strip.
Note: longer periods of abstinence = higher semen volume but lower
motility
5.3. Wait for the color of the impregnated zone to
become uniform (<30 seconds).
• Bladder should be evacuated prior to ejaculation 5.4. For normal samples, pH paper in the range 6.0 to
• Most satisfactory sample is when collection is done in the 10.0 should be used.
laboratory 6. Record the pH.
Note: if sample collected at home, delivered to laboratory within 1 hour

Microscopic examination of Semen
1. Making a wet preparation.

• Should not be collected in condoms
2. Examine the wet preparation using 100x magnification.
• Home sterilized containers should not be used
2.1. Take note of sperm aggregation, degree of
• Lid of the container should not have rubber lining
agglutination and point of attachment.
Note: water is spermicidal
Degree of agglutination
Grade 0 No adhesion
Sample Rejection:
Grade 1 Isolated (<10%)
Grade 2 Moderate (<30%)
• Unlabeled samples
Grade 3 Large (<50%)
• Incomplete ejaculation
• Sample received after 1 hour of ejaculation
• Inadequate transported sample Point of agglutination
Head-to-head
Sample Storage: Tail-to-tail Heads are seen to be free and
move clear of agglutinates
• Semen samples should be examined ASAP Tail-tip-to-tail-tip
• If delay, store at room temperature (200C - 300C) Mixed Clear head-to-head and tail-to-tail
agglutinations
• Excessive heat, cold, or direct sunlight should be avoided
Tangle Heads and tails enmeshed. Heads
• Do not refrigerate the samples are not clear of agglutinates as they
are in tail-to-tail agglutination.
Equipment and Reagents:
• Glass slide 2.2. Look for cells other than spermatozoa and estimate.
• Cover glass 3. Examine wet preparation using magnification x200 to
• Neubauer counting chamber x400.
• pH paper 3.1. Assess sperm motility.
• WBC pipette
• Cold water Sperm Motility Grading
• Timer Progressive (PR) Spermatozoa moving
actively, either linearly or in
• Microscope a large circle, regardless of
speed.
Macroscopic examination of Semen Non-progressive (NP) All other patterns of motility
with an absence of
1. Record the date and time of collection, medication taken, progression.
and time of analysis. Immotile (IM) No movement.
2. Record the liquefaction time.
Page 9 of 38
3.2. Look for spermatozoa in an area at least 5mm from
the edge of the coverslip.
3.3. Systematically scan the slide to avoid repeatedly
viewing the same area.
3.4. Start scoring a given field at a random distant. Scan
and count quickly.
3.5. Tally the number of spermatozoa in each motility
category.
3.6. Assess 200 spermatozoa / replicate.
4. Assess sperm number.
4.1. Aspirate semen up to 0.5 mark of WBC pipette.
4.2. Aspirate diluting fluid up to 11.0 mark.
4.3. Shake horizontally to mix.
4.4. Charge the hemocytometer chamber, stand for 10
minutes to allow immobilized sperm settle.
4.5. Count the sperm in 2 large squares (WBC squares).
4.6. Calculate for the sperm concentration based on the
following formula:
𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑥106 𝑝𝑒𝑟 𝑚𝐿) 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓
𝑠𝑝𝑒𝑟𝑚𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 100,000

= 133𝑥106 𝑝𝑒𝑟 𝑚𝐿 𝑥 100,000
= 13.3 𝑥106 𝑝𝑒𝑟 𝑚𝐿
4.7. To get the total sperm count, multiply the sperm

concentration by the volume of the whole ejaculate.
𝑡𝑜𝑡𝑎𝑙 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑢𝑛𝑡 = 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒
= 13.3𝑥106 𝑝𝑒𝑟 𝑚𝐿 𝑥 2𝑚𝐿
= 26.6 𝑥106 𝑝𝑒𝑟 𝑒𝑗𝑎𝑐𝑢𝑙𝑎𝑡𝑒
5. Record and print your result.
Page 10 of 38
MICROBIOLOGY Safety Precautions
CULTURE MEDIA PREPARATION • Use of PPE is required during the conduct of preparing
media.
Types of Media • Dehydrated culture media are highly hygroscopic. Keep
container tightly closed once opened.
According to Consistency: • Dehydrated culture media are for laboratory use only.
• Wash hands after every preparation.
1. Liquid (Broth) Media • Do not eat and use for food.
• Propagation of large number of organisms, fermentation • Observe aseptic techniques.
studies, and various other tests. • Do not use culture media after expiry date.
• Example: BHI Broth, Thioglycolate • Check the instruction label before storing the culture
media.
2. Semi-solid Media
• Cultivation of microaerophilic bacteria or for Basic Guidelines
determination of bacterial motility.
• Example: SIM, LIM 1. Storage
• Keep container tightly closed once opened.
3. Solid Media • Do not use culture media after expiry date.
• Allows media to grow in physically informative or useful • Check the instruction label before storing the culture
ways. media.
• Example: MHA, NA, BAP
2. Sterilization
According to Function: • Sterilization by heat is the most used.
• Media containing carbohydrates should not be
1. General (Basal / Basic) Media autoclaved at temperature exceeding 1160C to 1180C.
• Supports the growth of a wide variety of microorganism • In some liquid media, warming may lead to loss of activity
types and lacks inhibitory property. of some compound. In these cases, sterilization by
• Example: NA, TSA filtration must be performed.
2. Selective Media Process of Culture Media Preparation

• Favors the recovery of specific types or general of
microorganisms and inhibits other members of a mixed 1. Weigh an amount of the powder according to the
microflora. manufacturer’s instructions and place in an Erlenmeyer
• Example: MAC, TCBS, GBA, BCA flask.
2. Label the flask with the name of the medium and volume.
3. Differential / Indicator Media 3. Cover with aluminum foil.
• Possess certain ingredients that enable presumptive 4. Autoclave at 1210C for 15 minutes.
identification of a specific or species either form a pure or 5. After autoclaving, cool to 45-500C.
mixed culture. 6. For BAP, aseptically add 5-10% sterile defibrinated
• Example: MAC, TCBS sheep blood. Allow the blood to equilibrate at room
temperature before adding.
4. Enriched Media 7. Measure the blood in a sterile test tube or use a sterile
• Supports the growth of a wide variety of microorganism serological pipette or a graduated cylinder.
including some of the more fastidious ones. 8. Mix by gently swirling to avoid formation of bubbles.
• Example: BAP, CAP 9. Dispense / pour about 20mL into sterile petri dishes.
5. Enrichment Media Depth of agar layer must be 4mm.
• Used to increase the relative concentration of certain 10. Once solidified, label the plates with the name of the
microorganisms, including some of the more fastidious medium and preparation date.
ones. 11. Store the plates at 40C in an inverted position or for
longer shelf life, place in a sterile tight plastic bag (for up
• Example: Thioglycollate broth, Selenite F broth
to 2-3 months).
6. Transport Media
Quality Assurance
• For temporary storage of specimens being transported to
the laboratory for cultivation. Sterility Test
• Example: Cary Blair Transport Medium, Amies Transport
Medium
Page 11 of 38
1. Incubate two tubes or plates from each autoclaved or
filter-sterilized medium overnight at 36±10C.
2. Check tube or plate for growth after 24 hours of Blood
incubation.
3. Record quality control results in the designated When is a blood culture requested?
logsheets.
1. Acute illnesses
Culture Response Test / Growth Dependence Test 2. Fever of unknown origin
3. Acute infective endocarditis
1. For plated media: 4. Suspected bacterial endocarditis
1.1. Inoculate at least one strain to test for ability of a
media to support growth of the target pathogen. Components of a blood culture broth:
2. For biochemical media:
1. 1% Gelatin
2.1. Inoculate at least one organism that will produce a
a. Enhance growth of N. meningitidis
positive reaction and at least one organism that will
2. 0.1% Bacto Agar
produce a negative reaction.
a. Enhance growth of anaerobic organism
3. Use quality assurance organisms such as
3. 0.025% SPS
Staphylococcus aureus ATCC 25923, Escherichia coli
a. Anticoagulant
ATCC 25922 and Pseudomonas aeruginosa ATCC
b. Inhibits activity of complement and lysozyme
27853 or organism isolated from clinical specimens that
c. Detects phagocytosis
were previously well identified.
d. Inactivates therapeutic concentration of
4. Record quality control results as to whether the strain
aminoglycosides
used produces the appropriate biochemical reactions /
color on the test medium. Collection Method:
PROPER COLLECTION, TRANSPORT AND PROCESSING 1. Find puncture site. Clean site using 70% alcohol, followed
OF SPECIMENS by an iodine solution.
2. Sterilize the rubber stopper using alcohol swab.
How to successfully recover bacteria from clinical 3. Collect the required specimen amount of blood.
specimens? 4. Inoculate collected blood into the blood culture broth.
5. Label the specimen properly.
1. Advance planning
2. Collect appropriate and adequate amount of specimen Cerebrospinal Fluid
3. Proper packaging and transport
4. Ability of the laboratory to accurately perform the • 1st tube: protein and glucose and special tests
diagnostic test • 2nd tube: gram stain and culture
• 3rd tube: cell count and differential staining
General Guidelines
Effusions
1. Follow universal precaution guidelines.
2. Treat all specimens as potential biohazards. What and where to collect?
3. Collect specimen before antibiotic treatment.
4. Collect from appropriate site. 1. Synovial fluid: from joints
5. Practice proper and aseptic collection technique. 2. Pleural fluid: from pleural cavity (space between lungs
6. Ensure sufficient quantity. and inner chest wall)
7. Follow recommended time of transport. 3. Peritoneal fluid: from abdominal cavity
8. Label specimen accordingly. 4. Pericardial fluid: from pericardial cavity
9. Accompany specimen with complete request form. 5. Hydrocoele: from testes
Clinical Specimens for Aerobic Culture Upper Respiratory Tract
1. Sterile samples 1. Nasopharyngeal swab

a. Blood 2. Throat swab
b. CSF
c. Effusions Lower Respiratory Tract
2. Nonsterile samples
a. Upper respiratory tract specimens 1. Sputum
b. Lower respiratory tract specimens 2. Endotracheal aspirate
c. Urine 3. Bronchoalveolar lavage
d. Exudates
Page 12 of 38
Isolation Streak Technique
Suitability Criteria for Culture 1. The inoculation loop is dragged across the surface of the
agar back and forth in a zigzag motion until approximately
Classification of sputum based on leukocyte and squamous epithelial 30% of the plate has been covered.
cell densities
Cell numbers per x100 (low power) field 2. Sterilize the loop and rotate the plate 90 degrees. Starting
Group Leukocyte cells Epithelial in the previously streaked section, the loop is dragged
6 <25 <25
5 >25 <10 through it two to three times continuing the zigzag pattern.
4 >25 10-25 3. The procedure is then repeated once more until the 4th
3 >25 >25 quadrant being cautious not to touch the previously
2 10-25 >25
1 <10 >25 streaked area in order to get isolated colonies.
Note: Group 4-6: only sputum samples in these categories should be
cultured
Urine
Methods of Collection:
1. Midstream clean catch

2. Cystoscopy or Catheterization
3. Suprapubic aspiration
Continuous Streaking Technique (For urine only)
Transudates and Exudates
1. Get the loopful of the urine aseptically by submerging
1. Abscess vertically only the loop portion (shaft not included).
2. Ear / Eye discharge Inoculate the BAP by continuous streaking, spread the
3. Tissue urine downward from the 1st quadrant and streaking up to
the 4th quadrant without changing loop.
General rule on collection: Clean site first and disinfect surrounding area
before collecting sample.
Processing, Inoculation, Streaking and Incubation
Abscess Blood (Conventional Method)
Collection method: 1. Label the blood culture broth (BCB) with the accession
number of the sample (example: BL001). Incubate for 18-
• General: remove surface exudate by wiping with sterile 24 hours.
saline or 70% alcohol. 2. Subculture from BCB is one as follows:
• Open wound: aspirate or swab deep into the lesion to a. Label the plates with laboratory number of the
firmly sample the lesion’s “fresh border”. sample.
• Close wound: aspirate b. Mix the blood sample by swirling the blood
culture bottle 2-3 times.
Tissue
c. By using a forceps, get a sterile cotton ball with
70% alcohol and sterilize the rubber stopper
• Tissue collection is an invasive procedure and requires
than gently pass into the flame.
surgery by a trained physician.
d. With a sterile disposable syringe and needle
• Place the specimen in a sterile container on sterile gauze
aspirate at least 0.5mL blood from the bottle.
moistened with sterile saline.
e. Place a drop of the sample on each agar plate
• Transport to the laboratory. Do not refrigerate.
(BAP, CAP, and MAC).
• Tissue submitted in formalin is unacceptable for culture. f. Streak with isolation.
3. Incubate: BAP and CA, in 5-10% CO2 at 36±10C
Transport Time
incubator; MAC at 36±10C incubator, ambient air for 18-
Specimen Time recommended 24 hours.
Respiratory (sputum) 1 hour 4. Record the culture media plates used and the date and
Gastrointestinal (stool) 1 hour time of inoculation in the worksheet.
Blood 1 hour 5. If no growth, reincubate the BCB until 7 days at 36±10C,
CSF Immediately ambient air for further subculture.
Other body fluids Immediately
Urine 1 hour
Exudates and transudates 30 minutes
Plating Technique
Page 13 of 38
Culture Blood Agar Plate (BAP) Chocolate Agar Plate MacConkey Agar Blood Culture Broth (BCB)
media / (CAP) (MAC)
Enrichment
broth
Incubation 36±10C with 5-10% CO2 for 18-24 hours 36±10C for 18-24 hours
Remarks Subculture BCB after 24 hours, 72 hours, 5 days and 7 days onto BAP, CAP and MAC
Preliminary report: after 24 hours
Final report: after 7 days
CSF / Effusions 3. With a sterile Pasteur pipette, place one drop of CSF onto
the first quadrant of each plate (BAP, MAC, CA) and 3-5
1. Label the plates and tube with the accession number of drops in BHI broth.
the sample. 4. Streak with isolation onto each agar plate.
2. About 1mL preferred (If more than 1mL is submitted, 5. Incubate inverted plates: BAP and CA, in 5-10% CO2 at
centrifugation is recommended. Use the sediment [do not 36±10C; MAC at 36±10C, ambient air for 18-24 hours.
decant all fluid but leave at least 1mL]) for the 6. Record the culture media plates / tubes used and the date
bacteriological investigation. and time of inoculation in the worksheet.
Culture Blood Agar Plate (BAP) Chocolate Agar Plate MacConkey Agar Brain Heart Infusion (BHI)
media / (CAP) (MAC) Broth
Enrichment
broth
Remarks Preliminary report: after 24 hours
Wound Swab / Discharge 3. Submerge the swab in the thioglycolate broth then
aseptically break / cut the stick halfway.
1. Label the plates and tube with the accession number of 4. Perform isolation streak technique onto the BAP and
the sample. MAC.
2. Use 2 swabs. Using the 1st swab, roll the sides and tip of 5. Incubate inverted plates and thioglycolate broth at
the swab onto the upper corner of the 1st quadrant of BAP 36±10C for 18-24 hours.
and MAC. 6. Record the culture media plates / tubes used and the date
and time of inoculation in the worksheet.
Culture Blood Agar Plate (BAP) MacConkey Agar (MAC) Thioglycollate Broth (BCB)
media /
Enrichment
broth
Incubation 36±10C for 18-24 hours
Sputum / Endotracheal Aspirate 5. Incubate inverted plates: BAP, CA, GBA and BCA in 5-
10% CO2 at 36±10C; MAC at 36±10C, ambient air for 18-
1. Label the plates with the accession number of the 24 hours.
sample. 6. Record the culture media plates used and the date and
2. Get a loopful of purulent part of specimen to be tested and time of inoculation in the worksheet.
make an evenly thin smear on a slide for gram stain. Air
dry. (Before sterilizing the loop, dip the loop by rubbing in
sand alcohol jar to clean the debris left on the loop)
3. Using another sterilized loop, get another loopful of
purulent part of the specimen and inoculate each plate
with similar size and quality of sample onto the center of
the 1st quadrant.
4. Streak the isolation onto each agar plate.
Page 14 of 38
Culture Blood Agar Plate (BAP) Gentamicin Blood Agar / Bacitracin MacConkey Agar (MAC)
media / Chocolate Agar
Enrichment
broth
Throat Swab 3. Perform isolation streak technique.

4. Incubate inverted late in 5-10% CO2 at 36±10C for 18-24
1. Label the plates and tube with the accession number of hours.
the sample. 5. Record the culture media plates used and the date and
2. Roll the sides and tip of the swab onto the upper corner time of inoculation in the worksheet.
of the 1st quadrant of BAP.
Culture media / Enrichment Blood Agar Plate (BAP)
broth
Incubation 36±10C with 5-10% CO2 for 18-24 hours
Remarks Final report: after 2 days
Urine Inoculate the BAP by continuous streaking, spread the

urine downward from the 1st quadrant and streaking up to
1. Label the plates and tube with the accession number of 4th quadrant without changing loop.
the sample. 4. Use another sterile loop and get loopful of urine to
2. Mix thoroughly the urine specimen, if sample container is inoculate MAC agar and do the isolation technique.
small invert 3-4 times or swirl if the sample container is 5. Incubate inverted plates at 36±10C for 18-24 hours.
large. 6. Record the culture media plates used and the date and
3. Get the loopful of the urine aseptically by submerging time of inoculation in the worksheet.
vertically only the loop portion (shaft not included).
Culture media / Enrichment Blood Agar Plate (BAP) MacConkey Agar (MAC)
broth
Remarks Use calibrated loop either 10uL or 1uL for BAP inoculation and colony count. (Inoculate into
BAP by continuous streaking)
Stool / Rectal swab a. Label the plates (TCBS and SSA) with the
specimen number and the source (example:
1. Label the plates and tube with the accession number of S001 AP-TCBS, SOO1 SF-SSA).
the sample. b. For APW, aseptically get a loopful of broth at the
2. Dip the swab into the stool sample. surface and make an isolation streak onto
3. Inoculate the swab onto BAP, MAC, SSA, and TCBS. TCBS.
4. Inoculate by rubbing the swab onto the center of the 1 st c. Incubate the plates at 36±10C for 16-24 hours.
quadrant of one medium, changing sides of the swab d. For Selenite F, gently mix and get a loopful of
each time to another medium. Do not discard the swab broth and make an isolation streak onto SSA.
and proceed to the next step. e. Incubate the plates at 36±10C for 16-24 hours.
5. Dip the swab into the upper portion of the APW then into f. Record the culture media plates used and the
the Selenite F broth. date and time of inoculation in the worksheet.
6. Streak with isolation onto each agar plate.
7. Incubate inverted plates: BAP, MAC, TCBS, SSA plates, Culture Media / Incubation
at 36±10C in ambient air for 18-24 hours. Enrichment Broth
8. Incubate the Enrichment broth media: Blood Agar Plate 36±10C for 18-24 hours
a. At 36±10C in ambient air for 6 hours MacConkey Agar
b. If the processing of specimen was done in the Salmonella Shigella Agar
late office hours and no night duty, the Thiosulfate Citrate Bile
Salts Sucrose Agar
enrichment broth media should be subcultured
Selenite F Broth 36±10C for 6 hours or at
after and overnight of 16-18 hours incubation at Alkaline Peptone Water room temperature for 16-18
room temperature. hours
9. After incubation, subculture the enrichment broths Remarks:
After incubating the enrichment broths:
Page 15 of 38
1. For APW, aseptically get a loopful of broth at the IDENTIFICATION AND WORK-UPS OF SPECIMEN
surface and make an isolation streak onto TCBS.
2. For Selenite F, gently mix and get a loopful of broth and Blood
make an isolation streak onto SSA.
3. Incubate the plates at 36±10C for 18-24 hours After incubation, examine primary media plates (BAP, CA, and
Final report: after 2 days MAC) for growth.
1. Growth on BAP only
Describe the colony morphology: the size, shape, consistency (dry or moist); pigment; transparency / opacity; hemolysis (α, β,
or non-hemolytic)
Gram Stain
G (+) cocci Yeast cells
Catalase test Germ tube
(+) (-) ID
Coagulase test (slide / tube Optochin Report / Record
method) Bacitracin
CAMP
BE
6.5 NaCl
Arabinose
STY STY
Report / Record Report / Record
2. Growth on BAP and MAC are not the same, look also for other possible pathogens
specific for BAP).
Compare BAP and MAC. Check if the colonies are the same
and presence of pigment on each medium. (If other colonies
Describe the colonies on MAC (LF / LLF / NLF)
Describe if necessary the consistency as mucoid or dry
If mixed (different colony morphology) NLF or LF, describe also size, shape, consistency (dry or moist); pigment
Perform necessary biochemical tests
LF: Citrate, TSI, LIA, SIM, Urease LLF / NLF: Citrate, TSI, LIA, SIM, Urease, Dextrose, Maltose
STY
ID
Report / Record
3. Growth on CAP b. Reincubate the BCB until 7 days at 36±10C,

a. Describe colony morphology. ambient air for further subculture.
b. Perform gram stain. c. No growth after 7 days (final report).
c. G (-) bacilli or coccobacilli (pleomorphic) d. Record / Report.
d. X & V Factor Dependence Test Satellitism /
Hemolysis Test Wound Discharge / Swab
e. STY
f. ID After incubation, examine the primary media plates (BAP,
g. Report / Record. MAC) and Thioglycollate for growth.

4. No growth
a. Preliminary report.
• Quantity (light / moderate / moderately heavy / heavy growth)
• Describe the colony morphology: the size, shape, consistency (dry or moist), pigment, transparency / opacity, hemolysis
(α, β, or non-hemolytic)
Gram stain
(+) (-) ID
method) Bacitracin
CAMP
BE
6.5% NaCl
Arabinose
Page 16 of 38
STY STY
2. Growth on BAP and MAC 3. With evidence of growth on THIO but no growth on
primary plates
Compare BAP and MAC. Check if the colonies are the same a. Subculture to BAP and MAC.
and presence of pigment on each medium. (If other colonies b. Perform necessary work-up / STY.
are not the same, look also for other possible pathogens c. ID.
specific for BAP). d. Report / Record.
4. No growth on primary plates and THIO
a. Describe the colonies on MAC (LF / LLF / NLF). a. Preliminary report
b. Describe if necessary, the consistency as mucoid or dry. b. Reincubate thioglycolate broth until 4 days at
c. If mixed (different colony morphology) NLF or LF, 36±10C.
describe also size, shape, consistency (dry or moist), c. No growth after 4 days (final report).
pigment d. Report / Record.
d. Perform necessary biochemical tests
i. LF: Citrate, TSI, LIA, SIM, Urease CSF / Effusions
ii. LLF / NLF: Citrate, TSI, LIA, SIM, Urease,
Dextrose, Maltose After incubation examine the primary media plates (BAP, CAP
e. STY and MAC) and TSB / BHI growth.
f. ID
g. Report / Record. 1. Growth on BAP only

Gram stain
(+) (-) ID
method) Bacitracin
CAMP
BE
6.5% NaCl
Arabinose
STY STY
2. Growth on BAP and MAC

3. Growth on CAP
Compare BAP and MAC. Check if the colonies are the same a. Describe colony morphology.
and presence of pigment on each medium. (If other colonies b. Perform gram stain.
are not the same, look also for other possible pathogens c. G (-) bacilli or coccobacilli (pleomorphic).
specific for BAP). d. X & V Factor Dependence Test Satellitism /
Hemolysis Test
a. Describe the colonies on MAC (LF / LLF / NLF). e. STY
b. Describe if necessary, the consistency as mucoid or dry. f. ID
c. If mixed (different colony morphology) NLF or LF, g. Report / Record.
describe also size, shape, consistency (dry or moist), 4. With evidence of growth on THIO but no growth on
pigment primary plates
d. Perform necessary biochemical tests a. Subculture to BAP and MAC
i. LF: Citrate, TSI, LIA, SIM, Urease b. Perform work-up / STY
ii. LLF / NLF: Citrate, TSI, LIA, SIM, Urease, Dextrose, c. ID
Maltose d. Report / Record.
e. STY 5. No growth on primary plates and THIO
f. ID a. Preliminary report
g. Report / Record. b. Reincubate thioglycolate broth until 4 days at
36±10C.
Page 17 of 38
c. No growth after 4 days (final report). After incubation, examine the plates (BAP, MAC) for growth.
d. Report / Record. Quantitate growth on BAP by multiplying the number of each
colony type by 1,000 if 1uL (0.001mL) loop was used or by
Urine 100 if 10uL (0.01mL) loop was used.

Gram stain
(+) (-) ID
method) Bacitracin
CAMP
BE
6.5% NaCl
Arabinose
STY STY
2. Growth on BAP and MAC e. STY

f. ID
Compare BAP and MAC. Check if the colonies are the same g. Report / Record.
and presence of pigment on each medium. (If other colonies
are not the same, look also for other possible pathogens 3. No growth
specific for BAP). a. Reincubate plates up to 48 hours at 36±10C.
b. No growth at 48 hours (final report)
a. Describe the colonies on MAC (LF / LLF / NLF). c. Report / Record.
b. Describe if necessary, the consistency as mucoid or dry.
c. If mixed (different colony morphology) NLF or LF, Sputum / Endotracheal Aspirate
describe also size, shape, consistency (dry or moist),
pigment After incubation, examine the plates BAP, GBA, BCA, and
d. Perform necessary biochemical tests MAC) for growth.
i. LF: Citrate, TSI, LIA, SIM, Urease
ii. LLF / NLF: Citrate, TSI, LIA, SIM, Urease, 1. Growth on BAP only
Dextrose, Maltose
• Describe the colony morphology: the size, shape, consistency (dry or moist), pigment, transparency / opacity, hemolysis (α, β,
or non-hemolytic)
Gram stain
(+) (-) Germ tube
Coagulase test (slide / tube Optochin ID
method) Bacitracin
(+) (-) Sensitive Resistant Report / Record
STY Report / STY Report /
Record Record
Report / Report /
Record Record
2. Growth on BAP and MAC c. If mixed (different colony morphology) NLF or LF,
describe also size, shape, consistency (dry or moist),
Compare BAP and MAC. Check if the colonies are the same pigment
and presence of pigment on each medium. (If other colonies d. Perform necessary biochemical tests
are not the same, look also for other possible pathogens i. LF: Citrate, TSI, LIA, SIM, Urease
specific for BAP). ii. LLF / NLF: Citrate, TSI, LIA, SIM, Urease,
Dextrose, Maltose
a. Describe the colonies on MAC (LF / LLF / NLF). e. STY
b. Describe if necessary, the consistency as mucoid or dry. f. ID
Page 18 of 38
g. Report / Record.
3. Growth on BCA
a. Describe colony morphology.
b. Perform gram stain.
c. G (-) bacilli or coccobacilli (pleomorphic).
d. X & V Factor Dependence Test Satellitism /
Hemolysis Test
e. STY
f. ID
g. Report / Record.
4. Growth on BAP / GBA Triple Sugar Iron (TSI)
a. Look for suspicious Streptococcus pneumoniae
and describe the colony morphology, the size, • Used to differentiate among the different groups of
shape, consistency and α-hemolysis on BAP Enterobacteriaceae.
and GBA. Quantify. • Detects three primary characteristics of a bacterium:
b. Perform work-up for Streptococcus o The ability to ferment sugars
pneumoniae. o The ability to produce gas from the fermentation
c. STY. of sugars
d. Report / Record. o Production of large amounts of hydrogen sulfide
5. No growth • Procedure: stab through the center of the butt up to the
a. Reincubate plates up to 48 hours at 36±10C. bottom of the tube then draw out and from the lower
b. No growth at 48 hours (final report) portion of the slant, make a vertical streak then fishtail
c. Report / Record. over slant.
Stool
1. Observe for a typical colony on the following culture

media after 18-24 hours of incubation:
Culture Media Suspected Possible

Colonies Organisms
BAP White β- S. aureus, yeast
hemolytic / non-
hemolytic Lysine Iron Agar (LIA)
colonies
MAC Non-lactose Salmonella spp., • Used to determine whether a gram-negative rod
fermenter or Shigella spp.,
decarboxylates or deaminates lysine
colorless (NLF) Aeromonas spp.,
Plesiomonas • Lysine deamination occurs on the lysine slant
spp., E. coli • Lysine decarboxylation occurs in the lysine butt
TCBS Yellow or green Vibrio spp. • Procedure: swab twice through the center of the butt up
flat colonies (Y/G) to the bottom of the tube then draw out and from the lower
SSA NLF with or Salmonella spp., portion of the slant, make a vertical streak then fishtail
without H2S Shigella spp.
over slant.
2. Perform biochemical tests / serological typing on

suspected typica colonies on each medium
BIOCHEMICAL TESTS: IDENTIFICATION OF

FERMENTERS AND NON-FERMENTERS
Citrate
• Used to determine the ability of bacteria to utilize sodium

citrate as its only carbon source and inorganic ammonium
Sulfide-Indole-Motility Medium
dihydrogen phosphate (NH4H2PO4) is the sole fixed
nitrogen source. • A semi-solid medium used as differential test medium
• Procedure: Make a vertical streak then fishtail over slant. • Sulfide: detection of the ability of an organism to liberate
hydrogen sulfide (H2S) from sufur bearing amino acids
producing a visible, black color reaction
Page 19 of 38
• Indole: to determine the ability of an organism to split
indole from the tryptophan molecule by adding 3-5 drops
of Ehrlich’s or Kovac’s reagent and observe for
development of a red color
• Motility: to determine if the organism is motile or non-
motile
• Procedure: stab once through the center of the agar to a
depth of 1/3 to ½ of the medium making sure that the
inoculating needle was drawn in the same stab
• Indole: performed after incubation of the SIM O/F Maltose / Dextrose
• Tests the metabolism of sugar by prokaryotic cells. Cells

may metabolize sugar in a variety of pathways, and the
OF test is studying whether sugar is metabolized by
aerobic respiration or by an anaerobic pathway including
fermentation
• The OF medium has a low agar and peptone content, and
a high sugar content, making it a semi-solid medium that
is unlikely to go alkaline from protein utilization if the
sugars are metabolized
• Procedure: Inoculate the test medium by stabbing the
Urease medium 3x.
• Used to determine if the microorganism that possesses

the enzyme urease that can hydrolyze urea, releasing
ammonia and producing a pink, red color change in the
medium
• Procedure: inoculate the broth with a heavy inoculum
from an 18–24-hour pure culture
IDENTIFICATION OF STAPHYLOCOCCUS SPP.
• Gram-positive bacteria
• Round / cocci
• Grape-like clusters
• Non-spore forming bacteria
• Facultative anaerobes
• Heat-resistant organism
• Can tolerate high salt content media
Oxidase
1. Describe the colony morphology on BAP:
• Determine the presence of bacterial cytochrome oxidase • Size
using the oxidation of the substrate tetramethyl-p- • Color
phenylene dihydrochloride to indophenol, a dark, purple-
• Hemolysis (beta or gamma)
colored end product
• To separate Enterobacteriaceae from other bacteria like
Vibrios, Neisseria spp., Pseudomonas, Haemophilus,
and other related bacterial species
• Procedure:
1. Place a filter paper on a slide
2. Dispense 1 drop of reagent onto the filter
3. By using a sterile applicator stick, pick a colony from a
colorless media and rub on the moistened filter paper
Page 20 of 38
2. Gram stain
• Gram-positive cocci
• Form clusters “grape-like”
• Occur singly, in pairs, tetrads, short chains
Catalase Test
• Used to differentiate Staphylococcus (+) from

Streptococcus (-)
• Reagent: hydrogen peroxide (H2O2) – catalase mediates
the breakdown of H2O2 to hydrogen and water
• Results: Flow Diagram on Identification of Staphylococcus spp.
o Positive: immediate bubbling or effervescence
o Negative: no bubbling formed
• Procedure: Pick a colony from an 18–24-hour culture

and immerse into the H2O2. Do not mix.
Coagulase Test IDENTIFICATION OF STREPTOCOCCUS /

ENTEROCOCCUS
• Used to differentiate Staphylococcus aureus (positive)
from other Staphylococcus spp. (negative) – Coagulase Streptococcus spp.
Negative Staphylococcus (CoNS).
• Coagulase is an enzyme produced by S. aureus that • Gram-positive cocci in pairs and in chains
converts (soluble) fibrinogen in plasma to insoluble fibrin • Nonmotile, non-spore forming
• Facultatively anaerobic
1. Slide Coagulase Test • Catalase negative
• Screening • BAP and CAP
• Detects clumping factors (bound coagulase) • 350C in 5-10% CO2 atmosphere
• Read results in 10 seconds • Blood, CSF, Upper Respiratory Tract, Lower Respiratory
• Results: Tract, Exudates
o Positive: presence of white precipitate or
agglutination within 10-15 seconds Streptococcus Classification
o Negative: smooth and milky / homogenous
mixture • Hemolytic reaction on SBAP
2. Tube Coagulase Test • Physiologic – pyogenic, viridans, lactic and enterococcal
• Definitive • Lancefield – Group A-H, L-O
• Detects free coagulase
• Reagents: commercially prepared Rabbit’s plasma Laboratory Identification of Streptococcus spp.
/ dehydrated plasma containing citrate or EDTA
Bacitracin Susceptibility Test
• Examine tube after: 2 hrs, 4 hrs, and 24 hrs of
incubation • Used to distinguish Group A Streptococci, from other
• Results: Streptococci. This test is used to determine the effect of
o Positive: any degree of clotting / coagulum small amount of bacitracin (0.04U) on an organism.
o Negative: no clot / coagulum formation • Results:
o Susceptible: any zone of inhibition around the
disk
Page 21 of 38
o Resistant: no zone of inhibition • Identify Group B beta-hemolytic Streptococci (S.
• S. pyogenes: BACTRACIN SUSCEPTIBLE agalactiae) based on their formation of a substance
• Procedure: (CAMP factor) that enlarges the area of hemolysis formed
1. Create a three-layered on half of the BAP with a sterile by the beta-hemolysin elaborated from S. aureus.
loop containing few colonies if the test isolate. • Results:
2. Make sure that the lawn is formed side by side streaking o Positive: arrow-shaped zone of hemolysis
to ensure confluent growth. o Negative: no arrow-shaped hemolysis formed
3. With sterile forceps, place a bacitracin disk on the lawn. • Procedure:
Gently press the disk so that it adheres to the agar 1. Make a vertical line using a sterile loop with a few colonies
surface. of S. aureus ATCC 25923 on BAP.
4. Incubate the plate for 18-24 hours at 360C, 5-10% CO2. 2. Draw a perpendicular line of the test isolate about 4-6mm
away from the S. aureus ATCC 25923 line.
Bile Esculin
Optochin Susceptibility Test • Tests the ability of organisms to hydrolyze esculin in

the presence of bile. It is commonly used to identify
• Used in the presumptive identification of alpha-hemolytic members of the genus Enterococcus (E. faecalis and
Streptococcus pneumoniae, which is OPTOCHIN E. faecium)
SENSITIVE • Results:
• Results: o Positive: diffuse blackening of more than half of
the slant within 24-48 hours
Zone of inhibition Interpretation o Negative: no blackening
≥14mm Susceptible • Procedure: Inoculate the bile esculin medium with 2-
9 -13mm Intermediate 3 colonies of the test isolate with an inoculating
<9mm Resistant
needle.
• Procedure:
1. With a sterile loop, pick a single alpha-hemolytic colony
then create a three-layered lawn on half of the BAP.
2. Make sure that the lawn is formed side by side streaking
to ensure confluent growth.
3. Place an optochin disk on the lawn with a sterile forceps.
Gently press the disk for it to adhere to the agar surface.
4. Incubate the plate for 18-24 hours at 360C, 5-10% CO2.
6.5% NaCl
• Tests the ability of organisms to grown in high

concentrations of salt
• Results:
o Positive: turbidity of presence of obvious
bacterial growth in the medium
o Negative: clear or no growth
CAMP test • Procedure: Emulsify 2-3 colonies of the tests isolate
into 6.5% NaCl broth.
• Christie, Atkins, Munch-Petersen
Page 22 of 38
2. Satellitism / Hemolysis test
• When grow in blood culture media, S. aureus
produce NAD as a metabolic by product. For that
Arabinose Fermentation Test reason, species of Haemophilus may grow very
closely to the colonies of S. aureus when streaked
• Differentiates Enterococcus faecium (+) from on sheep blood agar.
Enterococcus faecalis (-) • S. aureus produces NAD (V factor), a key
• Results: requirement for the growth of H. influenzae. This
o Positive: yellow phenomenon is known as satelliting, and the test is
o Negative: no color change called satellitism.
• Procedure: Stab the tubed medium 5-8 times with 5- • Procedure:
10 colonies of the tests isolate. 1. Using the same suspension for the X and V
growth requirement tests, dip the sterile swab
IDENTIFICATION OF HAEMOPHILUS INFLUENZAE AND carefully into the bacterial suspension and
NEISSERIA GONORRHEAE streak in a close zigzag motion of BAP. Allow to
dry.
Haemophilus influenzae 2. Streak vertical line of Staphylococcus aureus on
the middle of the zizag pattern and incubate in a
• Large, colorless to gray colonies 5-10% CO2 incubator for 18-24 hours at 36+0C
• No discoloration of chocolate agar plate • Interpret as follows:
• With pungent odor 1. Positive: presence of growth with beta-
• Gram-negative coccobacilli hemolysis near or around the S. aureus streak
• Pleomorphic 2. Negative: presence of growth without beta-
• Growth requirements: hemolysis
o X factor or hemin
o V factor or NAD (nicotinamide adenine
dinucleotide)
Identification of Haemophilus influenzae
1. X and V factor Dependence test

• Determines nutrient requirement of organisms
• Procedure:
1. Prepare turbid suspension (1.0 McFarland)
2. Inoculate on trypticase soy agar plate
3. Place disks X, V and XV
Neisseria gonorrheae
4. Incubate in CO2 (+35 to +370C)
5. Results: • Grows on BAP (non-hemolytic) and CAP
• Gram-negative diplococci: coffee bean-shaped
• Can survive as an extracellular organism, or alternatively,
Haemophilus X V Beta- as an intracellular organism
species hemolysis
H. influenzae + + - Identification of Neisseria gonorrheae
H. parainfluenzae - + -
H. haemolyticus + + + 1. Oxidase test
H. - + + • Determines the presence of bacterial cytochrome
parahaemolyticus oxidase using the oxidation of the substrate
tetramethyl-p-phenylene dihydrochloride to
indophenol, dark, purple-colored end product
• N. gonorrheae: POSITIVE
2. Superoxol test
Page 23 of 38
• Catalase o Veillonella
• 30% hydrogen peroxide • All bacilli are gram-negative EXCEPT:
• Positive: effervescence o Bacillus
• Negative: little to no effervescence o Bifidobacterium
o Actinomyces
STAINING TECHNIQUES o Nocardia
o Streptomyces
Staining methods o Clostridium
o Corynebacterium
I. Positive Staining – actual cells are stained and appear o Erysipelothrix
in a clear background o Listeria
o Lactobacillus
1. Simple stain
• A stain which provides color contrast but gives same Morphology:
color to all bacteria and cells
• Example: Loeffler’s Methylene Blue
2. Differential stain
• A stain which imparts different colors to different
bacteria and cells
• Example: AFB stain
II. Negative Staining – the background is colored to create

a contrast to aid in the better visualization of cellular
structures
• Example: India Ink
Gram Stain
Direct from samples
• A differential staining technique based on bacterial
cell wall structure • Respiratory / Sputum samples
• Classifies bacteria as: o Suitability Criteria of Sputum for Culture:
o Gram-positive Classification of sputum based on leukocyte and
o Gram-negative squamous epithelial cell densities
Reagents: Low Power Field

Leukocytes / Pus cells Epithelial cells
• Primary stain: Crystal Violet Unfit for Culture:
• Mordant: Gram’s Iodine > 25 > 25
• Decolorizer: Acetone-Alcohol < 25 > 25
• Counter Stain: Safranin Red Fit for Culture:
> 25 < 25
Rules: < 25 < 25
• All cocci are gram-positive EXCEPT:

o Neisseria • Exudates / Urine / Tissue
o Moraxella
Pus cells, Epithelial cells / LPF Organisms / OIF
<1 / LPF 1+ RARE <1 / OIF 1+ RARE
1-10 / LPF 2+ FEW 1-10 / OIF 2+ FEW
11-25 / LPF 3+ MODERATE 11-25 / OIF 3+ MODERATE
>25 / LPF 4+ MANY >25 / OIF 4+ MANY
Note: Indicate the absence of cells as “NONE” and of organisms as “NO 2. Air dry and heat fix by passing the slide 2-3 times over a
ORGANISM SEEN”
flame and allow to cool.
3. Flood the smear with crystal violet and let stand for 1
Procedure:
minute.
1. Prepare a slide smear. Smear a very thin layer onto the 4. Wash gently with water. Pour off excess water.
slide, enough to dry completely within a few seconds. 5. Flood the smear with Gram’s iodine and let stand for 1
minute.
6. Wash gently with water. Pour off excess water.
Page 24 of 38
7. Decolorize with acetone-alcohol 5-10 seconds util the o Spot-early morning collection
alcohol runs most clear. Be careful not to decolorize. o Spot-spot collection (at least 1 hour apart)
8. Immediately wash with water. • Smear check points
9. Flood slide with safranin and let it stand for 1 minute. o Sputum quality
10. Wash gently with water. Air dry. o Staining
11. Examine under low power objective and estimate number o Cleanness
of squamous epithelial cells and leukocytes. o Thickness
12. Examine under oil immersion objective to determine o Size
predominant and other organisms present. o Stained smear has 2cm width and 3cm length
Quality Control Procedure:
✓ Control should be run on a daily basis 1. Using a wooden applicator stick, pick the purulent
✓ Standard color depends on the manufacturer, as long as particles of the specimen.
Gram-positive is recognizable from Gram-negative 2. Spread the specimen evenly unto the center of the slide
by making small, circular, coil-type pattern smear.
Important Points 3. Spreading should start on the innermost center to the
periphery border of the smear.
✓ Use fresh / young cultures – must be within 18-24 hours 4. Air dry and heat fix by passing the slide 2-3 times over a
old flame and allow to cool.
✓ Thin, thick or uneven smears will result in poor / uneven 5. Place fixed slide on the staining rack.
staining and decolorization 6. Pour carbol fuchsin on the smear covering it entirely.
✓ Do not heat-fix for very long 7. Apply enough heat underneath until steam comes off
✓ Perform quality control of reagents from the stain (Do not boil or dry out).
8. Leave it for 10 minutes.
India Ink
9. Gently rinse with tap water to remove excess carbol
• Staining of cerebrospinal fluid to test for the presence of fuchsin. At this point, the smear is red in color.
Cryptococcus spp. 10. Decolorize with acid alcohol until pink color disappears
from the smear.
Procedure: 11. Counterstain with Methylene blue for 2-3 seconds.
12. Gently wash with tap water and tip to drain excess water.
1. Place a drop of India ink on a clean glass slide.
2. Add a drop of sample to the India ink. Results
3. Using the edge of a cover slip, mix.
4. Cover the entire smear with the cover slip. Reporting scale AFB seen
5. Let it stand for 5 minutes. 0 No AFB seen in 300 visual fields
(VF)
6. Look for white cells in low power field then shift to high
+n 1-9 AFB seen in 100 VF (write the
power field. actual number of AFB seen)
1+ 10-9 AFB seen in 100 VF
Results 2+ 1-10 AFB / OIF in at least 50 VF
3+ More than 10 AFB / OIF in at least
Findings Reporting 20 VF
Encapsulated yeast cells – Positive for encapsulated
present yeast cell/s
Encapsulated yeast cells - No encapsulated yeast Other Tests
absent cells/s seen
Potassium Hydroxide (KOH) Mount
Acid-Fast Bacilli Staining • Used for the rapid detection of fungal elements in clinical
specimen, as it clears the specimen making fungal
• Rod-shaped bacilli that can be seen under the
elements more visible during direct microscopic
microscope following a staining procedure in which the
examination.
bacteria retain the color of the stain after an acid wash
• Reagent: 10% KOH
Direct Sputum Smear Microscopy (DSSM) • Procedure:
1. Place 1-2 drops of 10% KOH on a clean glass side.
• Involves the examination of a series of sputum specimens 2. Add a small amount of specimen in 10% KOH.
from each patient and requires repeated patient visits to 3. Mix the KOH and specimen using the edge of the cover
health facilities to submit specimens and to collect slip.
• Screening for pulmonary tuberculosis 4. Cover the drops with the cover slip.
• Pulmonary specimen (2 samples) 5. Wait for 5 minutes for cellular clearing.
Page 25 of 38
6. Examine the slide under the microscope with a reduced 2. Using a sterile applicator stick, pick several colonies form
light surface at LPO. the plate and emulsify on each drop, creating
7. Observe for the following structures: homogenous, slightly milky suspension.
• Yeast cells (budding or single cells) 3. Mix the anti-serum with the suspension using a sterile
• Pseudohyphae applicator stick.
• Hyphal elements: maybe hyaline (light or no 4. Tilt the glass slide back and forth for 1 minute and observe
pigment) or dematiaceous (dark brown) for agglutination.
8. Reporting: 5. Interpret as follows:
• Positive: report as “positive for (fungal elements • Positive: strong agglutination appears within 30
observed)” – yeast and hyphal elements seconds to 1 minute
• Negative: report as “no fungal elements found” • Negative: homogenous suspension
6. Agglutination is grossly observed with a light passing
KOH String Test through the slide. Delayed or weak agglutination is
regarded as negative.
• Relies on the differential resistance to 3% potassium
hydroxide between gram-positive and negative cells ANTIMICROBIAL SUSCPETIBILITY TESTING
• Results:
o Positive: organisms become thick, stringy and • Measures the ability of microbial agent/s to inhibit in vitro
form long strands within the first 30 seconds. bacterial growth
This is seen in gram-negative bacteria. • Indicated for an etiologic agent of an infection that needs
o Negative: organisms leave the suspension chemotherapy
unaltered or absence of stringing. This is seen • Guide the clinician in selecting the best and appropriate
in gram-positive bacteria. antimicrobial agent (to predict the outcome of treatment
• Procedure: with the antimicrobial tested)
1. Place a drop of 3% KOH on a clean glass side.
2. Emulsify a loopful of growth from a colony to the drop of Methods:
3% KOH.
3. Stir the suspension continuously for 60 seconds. 1. Disk Diffusion
4. Gently pull away the loop from the suspension. 2. Dilution Method
3. Gradient Diffusion Method
Germ Tube 4. Automated Antimicrobial Susceptibility Testing System
• An outgrowth produced by spores of spore-releasing Disk Diffusion

fungi during germination. The germ tube differentiates,
grows, and develops by mitosis to create somatic hyphae. • Antibiotic paper disks are placed on agar medium surface
• Procedure: inoculated with the test organism
1. Place 0.5mL of fresh serum in clean test tube. • Kirby-Bauer Method
2. Pick a colony then emulsify in the fresh serum.
Medium for Disk Diffusion
3. Incubate the suspension at 35-370C for 2-3 hours.
4. After incubation, place a drop of suspension on a clean
• Agar for AST
glass slide and cover with cover slip.
o pH 7.2-7.4
5. Examine under low and high-power objective.
o Low Thymine and Thymidine
6. Results:
o Sufficient divalent cation (Ca++ and Mg++)
• Positive: a short hyphal (filamentous) extension
• Mueller Hinton Agar
arising laterally from a yeast cells, with no
o Good batch to batch reproducibility
constriction at the point of origin. Germ tube is half
o Low in antibiotic inhibitors against sulfonamides
the width and 3-4 times the length of the yeast cell
trimethoprim, tetracycline
and there is no presence of nucleus.
o Supports the growth of most non-fastidious
• Negative: no hyphal (filamentous) extension arising
pathogens
from a yeast cell or a short hyphal extension
• AST Agars
constricted at the point of origin.
Organism Agar plate
Serotyping
Gram-negative organisms Mueller-Hinton Agar (MHA)
Staphylococcus spp.
• Serotypes are groups within a single species of Enterococcus spp.
microorganisms, such as bacteria or viruses, which share Streptococcus spp. Mueller-Hinton Agar with
distinctive surface structures. Neisseria mengitidis 5% sheep’s blood
• Procedure: Haemophilus spp. Haemophilus Test Medium
1. Place a drop of each anti-sera on a clean glass slide. (HTM)
Neisseria gonorrhoeae GC Agar
Page 26 of 38
Neisseria 9 3
gonorrhoeae
• Antibiotic disks Neisseria 5 2
o Stock antibiotic disks should be stored at -200C meningitidis
o Working antibiotic disks should be stored at 40C
o Antibiotic disks used are based on Clinical and
• Inoculation of Agar Plates
Laboratory Standards Institute (CLSI) panels
o Use appropriate check plate for a particular
• Disks per plate guide:
isolate
Organisms Maximum number of disks
Organism Check plate
140mm or 100mm or
150mm (big 90mm (small Gram-positive BAP
plate) plate) Staphylococcus spp.
Enterobacteriaceae 12 5 Streptococcus spp.
Pseudomonas Neisseria mengingtidis
aeruginosa Gram-negative MAC
Acinetobacter spp. Fastidious CAP
Enterococcus spp. Haemophilus spp.
Haemophilus spp. 9 4 Neisseria gonorrhoeae
Streptococcus 9 4
pneumoniae
Streptococcus spp. • Incubation Guide
Viridans group o Inverted plates are incubated at the desired
Streptococcus spp. temperature, atmosphere and duration of
Beta-hemolytic incubation depending on the test organism’s
group requirement
Organism Agar Temperature Atmosphere Time

Enterobacteriaceae MHA 350C ± 2 - 16-20h
Pseudomonas aeruginosa
Acinetobacter spp.
Burkholderia spp.
Stenotrophomas maltophilia 20-24 h
Other Non-Enterobacteriaceae 16-20 h
Staphylococcus spp. 24h
Enterococcus spp. 24h
Streptococcus spp. MHA with 5% 5% CO2 20-24h
Beta-hemolytic and Viridans group sheep blood
• 15-minute rules: within 15 minutes 8. With the same swab, inoculate by touching / rubbing the
o After preparing inoculum, seed the agar check plate, discard the swab accordingly.
o Apply the disk on seeded agar 9. Streak the inoculum in the check plate with sterile
o After disk application, incubate plate inoculating loop.
• Procedure: 10. Apply appropriate antibiotic disks manually by using
1. Label the MHA plate with the specimen accession sterile forceps onto the inoculated agar surface with a
number. minimum spacing of 24mm center to center between
2. Prepare 2-3mL sterile NSS in the sterile 5mL round disks.
bottom tube. 11. Gently press each disk down with sterile forceps for every
3. Using a sterilized inoculating loop, select and pick 3-5 application of the disk.
similar isolated colonies by touching the top of the • Reading Zones
colonies. o Using calipers or rule measure the diameter of
4. Mix the bacterial suspension by tapping or inverting the the complete zone of inhibition
tube. o Read MHA plates with unaided eye using
5. Using a densitometer, adjust the bacterial suspension to transmitted light
the required 0.5 McFarland standard. o On MHB, GC Agar, and HTM, remove the cover
6. Dip a sterile cotton swab into the standardized bacterial and measure inhibition zones from the surface
suspension, and express excess fluid against the inside illuminated with reflected light
wall of the tube.
7. Swab the entire surface of the AST medium 3x, rotating Assessing growth:
the plate through an angle of about 600C after each
application and pass the swab around the rim of the agar • Read plates only when the lawn of growth is confluent (A).
surface. • Repeat the test when individual colonies are apparent
(B).
Page 27 of 38
• Measuring zones of inhibition using transmitted light
a. For some tests (e.g., linezolid against
Staphylococcus spp.), measure zones of
Measuring the zones of inhibition: complete inhibition with transmitted light
b. Position the inverted petri plate in front of a light
• Measure zones of inhibition to the nearest whole
source for zone examination.
millimeter (mm).
• Trimethoprim-sulfamethoxazole
• Measure growth with no zone of inhibition as 6mm (Figure
a. When measuring the zone of inhibition for
B).
trimethoprim-sulfamethoxazole, disregard slight
• Zones of complete inhibition include the diameter of the or hazy growth (20% or less of the lawn of
disk and show no obvious, visible growth as judged by the growth) and measure the obvious zone margin.
unaided eye.
• Swarming
a. Strains of Proteus spp. may swarm into areas of
inhibited growth. Ignore the thin veil of swarming
growth in an otherwise obvious zone of
• Measure the zone of growth inhibition, not the zone of inhibition.
hemolysis. Tilt plate to better differentiate between
hemolysis and growth
. • Double zones
a. When double zones are observed, check the
• Discrete colonies growing within the zone growth for purity and repeat the test if necessary.
a. When discrete colonies grow within a clear zone b. If the culture is pure, measure the inner zone.
of inhibition, repeat the test with a pure culture
or subculture of a single colony from the primary
culture plate.
b. When the discrete colonies continue to grow
within the zone of inhibition after repeating the
test, measure the colony-free inner zone.
• Quality Control
o Antibiotics should be regularly tested if they still
function properly
o Guide to which antibiotic to test, with what
organism, and their range are indicated in the
CLSI Document no. M100
o Control strains MUST be used as reference:
Page 28 of 38
▪ Staphylococcus aureus 25923 2. mec-A-mediated Oxacillin Resistance using Cefoxitin
▪ Escherichia coli 25922 3. Inducible Clindamycin Resistance
▪ Pseudomonas aeruginosa 27853 III. For inducible Clindamycin Resistance (ICR)
▪ Streptococcus pneumoniae 49619 1. Staphylococcus spp.
▪ Haemophilus influenzae 49247 2. Beta-hemolytic Streptococcus spp.
▪ Escherichia coli 35218 3. Streptococcus pneumoniae
Minimal Inhibitory Concentration Tests recommendations for Enterobacteriaceae
• Lowest concentration of an antimicrobial agent that would 1. Extended spectrum beta-lactamase test (ESBL)
inhibit visible in vitro growth of a test organism over a a. E. coli / Klebsiella spp. / P. mirabilis
defined interval related to the organism’s growth rate 2. AmpC beta-lactamase screening test (not yet
• Determined by: recommended by CLSI)
o Dilution method 3. Test for diminished fluroquinolone susceptibility using
o Gradient diffusion method pefloxacin (extraintestinal Salmonella)
o Automated antimicrobial susceptibility testing 4. Carbapenemase screening test
system 5. Metallo-beta-lactamase test (MBL)
DETECTION OF MULTIPLE DRUG-RESISTANT Methicillin-resistant Staphylococcus aureus (MRSA)

ORGANISMS (MDRO)
• mecA or mecC-mediated oxacillin resistance in
• Bacteria that have become resistant to more than one Staphylococci
class of antimicrobial agents • Disk diffusion: Cefoxitin as surrogate antibiotic
• Usually are resistant to all but one or two commercially • Medium:
available antimicrobial agents, complicating treatment of o MHA with 4% NaCl for oxacillin only
illnesses they cause o MHA without NaCl for cefoxitin and other
antibiotics
Test recommendations for different organisms: • Procedure:
1. Following the standard disk diffusion, place cefoxitin disk
I. Direct beta-Lactamase Test (Nitrocefin disk) for: onto plain MHA. (MIC: Oxacillin onto MHA with 2-4%
1.Enterococcus spp. NaCl).
2.Haemophilus influenzae / parainfluenzae 2. Incubate plain MHA (cefoxitin) for 16-20 hours.
3.Moraxella catarrhalis 3. Read the susceptibility results.
4.Neisseria gonorrhoeae 4. Note: for oxacillin (and vancomycin), read with
5.Neisseria meningitidis transmitted light; for cefoxitin (and other antibiotics), read
6.Staphylococcus spp. with reflected light.
7.Pasteurella spp. 5. Record and interpret according to the latest CLSI
II. Screening for Staphylococcus spp. document.
1. Direct beta-Lactamase Test
mecA-mediated Cefoxitin disk Oxacillin MIC Oxacillin report Interpretation
resistance
mecA-mediated (or the R R R (or do not report) MRSA
rare mecC)
mecA-homologues (e.g., R R R (or do not report) MRS (other CoNS spp.)
mecA1, mecA2, mecC1)
(-) S R R Not MRSA / MRS
(-) S S S MSSA / MSS
Penicillin Disk Zone Edge Test for beta-Lactamase R (+) Sharp R

Production of Staphylococcus spp. S (-) Fuzzy S
>29mm
• Performed on MHA using a 10-U penicillin disk following S (+) Sharp R
CLSI guidelines >29mm
D-Test: Inducible Clindamycin Resistance (ICR)
• After 16-16 hours of incubation in ambient air, a sharp
zone edge was interpreted as positive and a fuzzy zone
• A disk diffusion test using clindamycin and erythromycin
as negative for beta-lactamase production
disks placed in close proximity to detect the presence of
inducible clindamycin resistance in Staphylococci and
Penicillin Beta- Penicillin Penicillin
disk lactamase disk zone report Streptococci.
test report edge test • Procedure:
Page 29 of 38
1. Following the general procedure for antimicrobial spp., K. aerogenes, Citrobacter spp., Serratia spp., and
susceptibility testing, place clindamycin 2ug disk (15- some other gram-negative species.
22mm, Staphylococcus spp.) apart from edge of • Procedure:
erythromycin 15ug disk on the susceptibility plate. 1. Following the general procedure for antimicrobial
susceptibility testing, place Cefotaxime (30ug) disk 15-
Note:
25mm apart from edge of imipenem 10ug disk on the
susceptibility plate.
✓ Beta-hemolytic Streptococcus spp. (12-15mm disk distance)
✓ Streptococcus pneumoniae (12-15mm disk distance) 2. Incubate plates for 16-24 hours.
3. Read susceptibility test results and observe presence of
2. Incubate plates for 16-24 hours. phenotypic resistance.
3. Read susceptibility test results and observe 4. Positive: flattened edge of the inhibitory zone around
presence of ICR. Cefotaxime disk adjacent to an Imipenem disk.
4. Positive: flattening (D-zone) of clindamycin zone 5. Negative: no flattened edge of the inhibitory zone around
adjacent to Erythromycin disk or hazy growth inside Cefotaxime disk adjacent to an Imipenem disk.
the zone of inhibition (no D-zone) adjacent to
erythromycin disk.
5. Negative: no flattening or hazy growth of
clindamycin zone adjacent to erythromycin disk.
Extended Spectrum beta-Lactamase
• Plasmid-mediated enzyme produced by

Enterobacteriaceae derived from mutations of TEM-1,
TEM-2, and SHV-1
• Capable of hydrolysing extended spectrum
cephalosporins, penicillins, and aztreonam
• Most often associated with: Carbapenemase
o E. coli
o Klebsiella spp. • Beta-Lactamases with versatile hydrolytic capacities.
• Note: routine screening of Proteus mirabilis for ESBL They have the ability to hydrolyze penicillins,
production is not recommended. However, when it is cephalosporins, monobactams, and carbapenems.
deemed clinically relevant, (ex., Bacteremic isolate) Bacteria producing these beta-lactamases may cause
screen for ESBL. serious infections in which the carbapenemase activity
• Can be transferred to enteric bacilli (Salmonella, Shigella, renders many beta-lactams ineffective.
Citrobacter freundii and Serratia marcsens) • Carbapenemase activity in Enterobacteriaceae and P.
• Appear to susceptible to certain antibiotics aeruginosa can be confirmed using the CarbaNP
• ESBL producing isolates are susceptible to beta-lactam / colorimetric microtube assay or the modified carbapenem
beta-lactamase inhibitor combination agents in vitro but inactiviation method (mCIM) test.
the effectiveness is uncertain • Both the CarbaNP and mCIM tests may detect
o Example: Clavulanic acid, Sulbactam, carbapenemase production, but neither of these tests can
Tazobactam: inhibit the ESBLs produced by the identify which carbapenemase is present.
organisms when tested with cefotaxime, • Procedure:
ceftazifime, cefepime or aztreonam) 1. For each isolate to be tested, emulsify a 1-uL loopful of
• Double Disk Diffusion Method bacteria for Enterobacteriaceae or 10-uL loopful of
o Detection of ESBL is by using a disk containing bacteria for P. aeruginosa from an overnight blood agar
clavulanic acid (AMC) and placing it between plate in 2mL TSB.
cefotaxime (CTX) and aztreonam (ATM) disk. 2. Vortex or mix by inversion for 10-15 seconds.
o An elliptical clearing of distortion of inhibition 3. Add a 10-ug meropenem disk to each tube using sterile
between the 2 disks indicates inhibition of beta- forceps or a single disk dispenser. Ensure the entire disk
lactamase by clavulanic acid is immersed in the suspension.
o Screening test 4. Incubate at 350C ± 20C in ambient air for 4 hours ± 15
minutes.
AmpC beta-Lactamase 5. Just before or immediately following completion of the
TSB-Meropenem disk suspension incubation, prepare a
• Chromosomal or plasmid-encoded enzymes. Isolates 0.5 McFarland suspension (using the colony suspension
that produce AmpC enzymes may have antimicrobial method) of E. coli ATCC 25922 in nutrient broth or saline.
susceptibility profile similar to those that produce ESBLs 6. Inoculate an MHA plate with E. coli ATCC 25922 as for
in that they show reduced susceptibility to penicillin, the routine disk diffusion procedure making sure the
cephalosporins, and aztreonam found in Enterobacter inoculum suspension preparation and MHA plate
Page 30 of 38
inoculation steps are each completed within 15 minutes. o If the test isolate produces a carbapenemase,
Allow the plates to dry for 3-10 minutes before adding the the meropenem in the disk will be hydrolyzed
Meropenem disks. and there will be no inhibition or limited growth
7. Remove the Meropenem disk from each TSB- inhibition of the meropenem-suscpetible E. coli
Meropenem disk suspension using a 10-uL loop of ATCC 25922.
placing the flat side of the loop against the flat edge of the • Carbapenemase negative:
disk and using surface tension to pull the disk out of the o Zone diameter of ≥19mm (clear zone).
liquid. Carefully drag and press the loop along the inside o If the test isolate does not produce
edge of the tube to expel excess liquid from the disk. carbapenemase, the meropenem in the disk will
Continue using the loop to remove the disk from the tube not hydrolyzed and will inhibit growth of the
and then place it on the MHA plate previously inoculated meropenem-suscpetible E. coli ATCC 25922.
with the meropenem-susceptible E. coli ATCC 25922 • Carbapenemase indeterminate:
indicator strain. Disk capacity: 4 disks on a 100mm MHA o Zone diameter of 16-188mm.
plate; 8 disks on a 150mm MHA plate. o Zone dimeter of ≥19mm and the presence of
8. Invert and incubate the MHA plates at 350C ± 20C in pinpoint colonies within the zone.
ambient air for 18-24 hours. o The presence or absence of a carbapenemase
9. Following incubation, measure the zones of inhibition as cannot be confirmed.
for the routine disk diffusion method.
10. Interpret as follows: REPORTING OF CULTURE RESULTS
• Carbapenamase positive:
o Zone diameter of 6-15mm or presence of
pinpoint colonies within a 16-18mm zone.
Specimen Preliminary Report Final Report Clinical Significance Other comments
Blood The preliminary report Final report with the If a typical pathogen is Record both preliminary and
for the absence of presence of growth should found from blood final report and date of
growth should be be reported as: “POSITIVE culture it is almost release in the designated
reported as “NO FOR (IDENTIFIED always significant. But logbook.
GROWTH AFTER 24 ORGANISM) AFTER many bacteria are
HOURS OF (HOURS / DAYS) OF often significant but
INCUBATION”. INCUBATION”. may occur as
(Susceptible test results contaminants in blood
included if applicable) culture. Therefore, the
finding may be
Final report with negative discussed with the
results for any organisms clinician or place a note
should be reported as: in the results from
“NO GROWTH AFTER 7 “Please correlate
DAYS OF INCUBATION”. clinically”.
CSF / Effusions The preliminary report Final report with the If a typical pathogen is Culture examinations:
for the absence of presence of growth on found from CSF culture
growth should be both primary plates and it is almost always Absence of colonies on the
reported as “NO BHI should be reported as: significant. But many primary plates (direct
GROWTH AFTER 24 “POSITIVE FOR bacteria are often inoculation of specimen onto
HOURS OF (IDENTIFIED significant but may agar plates) and BHI broth
INCUBATION”. ORGANISM)”. occur as contaminants has no sign of growth, record
(Susceptible test results in CSF culture. and report. Primary plates
included if applicable) Therefore, the finding and BHI broth are inspected
may be discussed with everyday for the sign of
Final report with presence the clinician or place a growth.
of growth on BHI only note in the results from
should be reported as: “Please correlate Re-incubate all “No growth”
“NO GROWTH ON clinically”. plates up to 2 days (after
PRIMARY ISOLATION. reading all the plates, re-
POSITIVE FOR incubate).
(IDENTIFIED
ORGANISM) (FROM BHI Perform subculture if BHI
BROTH AFTER DAYS OF broth has sign of growth
INCUBATION)”. while there are no growth on
(Susceptible test results primary plates.
included if applicable)
Page 31 of 38
Final report with negative If primary plates and BHI
results for any organisms broth are positive, correlate
should be reported as: the organisms isolated.
“NO GROWTH AFTER 4
DAYS OF INCUBATION”.
Record both preliminary and

final report and date of
release in the designated
logbook.
Exudates The preliminary report Final report with the Record both preliminary and
for the absence of presence of growth on final report and date of
growth should be both primary plates and release in the designated
reported as “NO thioglycolate should be logbook.
GROWTH AFTER 24 reported as: “POSITIVE
HOURS OF FOR (IDENTIFIED
INCUBATION”. ORGANISM)”.
(Susceptible test results
Final report with presence

of growth on thioglycolate
only should be reported
as: “NO GROWTH ON
PRIMARY ISOLATION.
POSITIVE FOR
(IDENTIFIED
ORGANISM) (FROM
THIOGLYCOLATE
BROTH AFTER DAYS OF
INCUBATION)”.
(Susceptible test results
Final report with negative

results for any organisms
should be reported as:
“NO GROWTH AFTER 4
DAYS OF INCUBATION”.
Urine Preliminary report is Presence of growth: Three or >3 differenct

not routinely done “(QUANTITY) CFU OF colonies should be
after 24 hours of (IDENTIFIED considered and reported as
incubation for the ORGANISM) PER ML OF “MIXED CULTURE” and
presence and URINE”. (Susceptible test suggest a “REPEAT
absence of growth. results included if COLLECTION”.
applicable)
Three or >3 different
Absence of growth: “NO colonies should be
GROWTH AFTER 48 considered if the patient is
HOURS OF an emergency case or in
INCUBATION”. catheter. Identify and
perform susceptibility testing
on predominant organisms
and the colony count is
>100,000.
Record both preliminary and

final report and date of
release in the designated
logbook.
Page 32 of 38
Stool Preliminary report is Final report with presence Record both preliminary and
not routinely done. of growth on both primary final report and date of
plates and BHI should be release in the designated
reported as: “POSITIVE logbook.
FOR (IDENTIFIED
ORGANISM)”.
Stool specimen is known

to have normal bacterial
fecal flora. If no suspected
pathogens observed or
tested, report: “NO
IMPORTANT
ENTEROPATHOGEN
ISOLATED”.
If no colonies found in
culture, report: “NO
GROWTH AFTER 2 DAYS
OF INCUBATION”.
Page 33 of 38
HISTOPATHOLOGY
• Branch of biology which deals with the study of diseased

tissue under microscope.
• Greek words:
o Histo = Tissue
o patho = Disease
o -logy = Study
• Biopsy: removing of bit of tissue from living-being
• Autopsy: removing of large tissue / organ from deady
body Sample Collection and Preservation
• Autolysis: destruction of tissue due to its own enzyme
• Putrefaction: destruction of tissue due to environmental • Tissue are collected and fixed immediately in a 10%
bacteria and microorganism neutral buffered formalin. Formalin causes:
o Chemical and physical changes
Purpose of Histopathology: o Hardens and preserve the tissue
o Protect from degeneration, autolysis and
1. Diagnose stage of cancer putrefaction
2. Diagnose pre-cancer stage • Ideally, there is at least 3 times the volume of fixative in
3. Other inflammatory diseases ratio to the size of the specimen (1:3) to ensure adequate
fixation.
• Pathologists understand between the normal and • In some other books, fixative is 10-20 times greater than
abnormal tissues based on their: the size of tissue.
o Size • Tagging of specimen is highly recommended for easy
o Cell shape orientation and recognition of the tissue.
o Nucleus to cytoplasm ratio o Every institution should evolve tagging
• In a normal condition, cytoplasm is bigger than nucleus mechanism. For example:
while in abnormal condition (cancer cells), cytoplasm is ▪ 1 tie for right fallopian tube to easily
shorter than nucleus. identify it with left fallopian tube.
o This should be clearly defined in the
Flow Chart for Histopathology
histopathology request form.
START: Operating Room • Large radical specimen resection should not be sliced /
cut by the surgeon but should be sent directly to the
1. Sample collection: histopathology department.
a. Accurate patient identification o Loafing: cutting the specimen into 3 or more
b. Orientation of samples parts so that fixative can easily penetrate the
c. Adequate fixation tissue. This applies to large radical specimen
2. Sample receiving: such as Modified Radical Mastectomy (MRM)
a. Specimen acceptability and Total Abdominal Hysterectomy and Bilateral
b. Completeness of request Salphingo Oophorectomy (TAH-BSO).
3. Entering specimen details • Small fragile specimen such as punch biopsy and gastric
4. Requested procedure charging (Histology, Laboratory tissues can be wrapped in a gauze envelope so they do
clerk, Medical Technology staff) not disintegrate during transport.
5. Process of specimen (Medical Technology staff, • Test requisition form for histopathology and
Histotech) cytopathology:
a. Proper embedding technique o Patient demographics
b. Microtomy o Operation
c. Staining o Type/s of specimen
d. Avoiding unacceptable artifacts o Number of specimen and container submitted
6. Finally (Pathologist) o Details of history (clinical data)
a. Inspection of controls o Clinical impression
b. Slides reporting
Sample Transportation and Accessioning
• Transported at room temperature and must be labeled

with:
o Complete patient’s name
Page 34 of 38
o Date and time collected 2. Cutting of specimen on both ends and one tangential cut
o Type/s of specimen (combined longitudinal and crosscut). Cut specimen at no
• Well-sealed, look-prof container more than the thickness of 5mm. = BLOCK 3
• Must not be place into any other solution or dry container 3. Get the tissue cassette and place the tissue on it.
except for frozen section request as irreversible 4. Cover the tissue cassette.
deterioration will take place making accurate microscopic 5. Label with corresponding accession number.
interpretation impossible.
• One of the biggest challenges histopathology facing is to TISSUE PROCESSING
accurately label and tract specimens to avoid potential
misidentification errors. • Hardens the tissue, enough to enable cutting into 3-5
• Patient and specimen identification are critical elements micron sections which then can be stained and examined
in surgical pathology. under a microscope.
• Proper identification of specimen by instructing • Once the tissue is properly fixed, it goes through a
technicians to use at least two patient identifiers when process which involves the following steps:
receiving samples o Dehydration
• Specimen identification is maintained across step like: o Clearing
o Specimen labeling o Infiltration / Impregnation
o Grossing • Water should be removed from the tissue and
o Block labeling progressively replaced by wax, to make a tissue block
o Slide labeling suitable for sectioning.
• Dehydration: removal of water from aqueous-fixed tissue
GROSSING and progressively immersed with ascending
concentration of alcohol.
• Precise and systematic gross description must be jot • Concentration used:
down which includes: o 3 levels of 95% alcohol (each level for 1 hour)
o Color o 3 levels of 100% alcohol (each level for 1 hour)
o Size • Water is driven out but still alcohol and wax are
o Consistency immiscible (can not be mixed) so we need a reagent that
o Shape can mix both and can act as a bridge between the two =
• Dissection and selection of sections for microscopic study ORGANIC SOLVENT XYLENE
are crucial parts of the pathologic examinations. • Clearing: replacing a dehydrant with substance that will
• Should be done by a Pathologist and may be assisted by be miscible with the embedding medium
Medical Technologist or Histotechnician. • Chase out the xylene and immerse the tissue in molten
• Cartilaginous, bony and hard tissues are placed in the wax at 600C.
decalcifying solution for 1-7 days. • Impregnation / Infiltration: final xylene is replaced with
• Number of bits received must be noted especially in small molten ax which infiltrate the tissue
biopsies (punch, core needles, and gastric biopsies). • Tissue processing is routinely done on an instrument
• Representative samples of the tissues are placed in the called Automated Tissue Processor
tissue cassettes using a scalpel and fine pointed forceps
and each tissue cassettes are labeled with accession Automated Tissue Processing (Leica TP 1020 Tissue
number. Processor)
• Inking of samples is integrated into the grossing system
• Leica TP1020 Tissue Processor: a carousel type semi-
to evaluate the margin of resection on microscopy.
enclosed tissue processor with 12 stations
• After grossing, all instrument are rinsed with water and
• It is the perfect combination of proven technology and a
wiped clean to prevent any mixed up floaters of tissues.
modern, functionally enhanced design.
• It is ideal to perform grossing:
• Gentle specimen processing and maximum safety at all
o In a well-ventilated, well-lit grossing stations
stages of processing are the result of robust engineering
o Exhaust and proper light are switched on
design based on proven and precise mechanics in
o Proper gloves mask and apron should be worn
conjunction with a modern user interface.
Procedure of Grossing (Appendectomy): • The user-friendly, easy-to-use control panel has buttons
that are arranged in functional groups.
1. Jot down the following description: • The easy-to-read LCD indicates the station parameters
• Color: grayish brown such as number of tissue baskets, vacuum function and
• Shape: tubular remaining infiltration time, real time, start time (delayed
• Consistency: soft to rubbery start), overall duration and end of run time.
• Measurement: LxWxH (4.0cm x 1.0cm x 0.5cm) • The specimen throughput can be doubled by using a
second tissue basket for improved productivity in routine
and research laboratories.
Page 35 of 38
• The tissue basket is moved up and down in the liquid at Embedding
three-second intervals to ensure thorough and even
mixing of the reagents and optimum infiltration of the 1. One cassette at a time to maintain specimen integrity.
tissue. 2. Choose the appropriate metal mold keeping aware of the
• Sealing rings on the container lids reduce solution loss size restriction of the tissue.
and therefore also mission into the ambient atmosphere 3. Orient the tissue correctly. Example: tissue to be on edge,
to a minimum. All reagent stations on the Leica TP1020 on end, or epithelium facing same direction
tissue processor are easily accessible because the 4. Place cassette top over the mold and fill with paraffin.
instrument can be rotated nearly immediately and without 5. Oven for 1 hour at 700C.
difficulty by using the integrated and adjustable rollers. 6. Set on to the cold plate neatly and in order for 30 minutes.
• The tissue specimens are protected from drying out even 7. Remove the block from the mold.
during a power failure since the tissue baskets are 8. Clean the block of excess paraffin.
automatically immersed in a station. The program is
Microtomy (section cutting)
resumed where interrupted once main power is restored.
• After a long-term power failure, the wax will be liquified. If
Microtome: instrument used to cut section from tissue embedded in paraffin
the programmed infiltration time for any of the stations is wax
exceeded a warning message is displayed indicating the
station number and the time in excess of program. 1. Use the lever to secure the tissue block
• The Leica TP1020 tissue processor vacuum can be 2. The hand wheel is use to move the block downward
applied to any of the stations both in manual and across the blade.
automatic operation. The advantage is it substantially 3. Safety knob must be turned downward, and the handle
improved infiltration of tissue in a shorter time. pulled outward.
Instruments with the vacuum feature are equipped with 4. The blade should be replaced or adjusted before you cut.
anodized aluminum containers. Use new tissue block and make sure to be careful when
• 1st station: 10% Neutral Buffered Formalin handling the razor blades.
• 2nd station: 95% alcohol 5. When it needs to be cleaned, use only a brush on an
• 3rd station: 95% alcohol upward motion.
• 4th station: 95% alcohol • Front knob is used to move forward and backward.
• 5th station: 100% alcohol • Mabhab is used for lateral adjustments.
• 6th station: 100% alcohol • Knife angle knob changes the angle of knife holder.
• 7th station: 100% alcohol 6. Cutting works best when the knife holder is set to a fairly
• 8th station: xylene steep angle.
• 9th station: xylene 7. Remove the outer layer of wax in the block (trimming).
• 10th station: xylene 8. Adjust the knife holder as close as possible to the tissue
• 11th station: melted paraffin wax block.
9. Set the thickness indicator to 5 microns.
• 12th station: melted paraffin wax
10. Remove and place on ice bath.
• Advantages:
11. Begin to cut and make a ribbon of 6.
o Reduces training time. Intuitive function
12. Place the ribbon on a warm water bath at 450C.
means it is easy to learn to how to use, which
13. Two of the ribbon strings will go to the slide.
reduces training time.
14. Label the corresponding accession number and place in
o Variable workloads. 100 or 200 cassettes
the staining rack.
processing at a time supports variable
workloads.
De-paraffinization (removing of excess paraffin wax)
o High reliability. Proven “workhorse” track
record of high reliability. 1. Incubate the slide for 30 minutes at 700C.
o Mechanical operation. Possible mechanical 2. Place in 2 levels of xylene (5 minutes each level).
operation during power outages keeps 3. Five times dips of 95% ethyl alcohol.
specimens from drying out. 4. Ten dips in running water.
EMBEDDING, MICROTOMY, DEPARAFFINIZATION Staining (hematoxylin and eosin)
• We need the following: 1. Hematoxylin stain for 20 minutes.

o Paraffin wax 2. 10 times dip in running water.
o Gloves 3. 1 dip in 1% acid alcohol.
o Metal molds 4. 10 dips in running water.
o Forceps 5. 2 dips in ammonia water.
o Tissue cassettes 6. 5 dips in running water.
7. 10 dips in eosin.
8. 10 dips in 95% ethyl alcohol.
Page 36 of 38
9. Another 10 dips in 95% ethyl alcohol. normal component of the squamous epithelial)
10. 2 levels of xylene for 2 minutes each level. in ORANGE
11. Blow dry the slides. o Eosin Azure-50: second counter stain; different
formulation with a number, according to the
Mounting proportion of the constituent dye; commonly
used; polychromatic stain (mixture of 3 stains)
1. Apply protective cover slip. o Eosin Y: stains the cytoplasmic of cells in
2. Send the slides to the pathologist for diagnostic shades of PINK or GREEN, depending on the
examination. pH.
THE SCIENCE OF TISSUE PROCESSING Principles and Procedures
• Prior to slicing, all specimens must be processed. 1. Fixation

• This includes dehydrating the specimen, introducing a • Fixed with 100% alcohol, 95% ethyl alcohol or spray
clearing agent, and embedding the specimen in paraffin alcohol fixative.
wax. • Stand for 20 minutes
• The specimen is cooled in an ice bath which helps the 2. Nuclear staining (Hematoxylin)
specimen be sliced more easily. • Using Harris Haematoxylin for 5 minutes
• The Histotechnician uses a microtome machine to slice • Nucleus is deliberately over stained
the tissue into small ribbons. 3. Differentiation (1% Acid Alcohol)
• Typically, at a thickness of 3-5 microns, less than the • Take off additional hematoxylin stain
thickness of a single cell. • As often results of hypochromasia
• The ribbons are placed in a warm water bath which helps • Contrast between counter stain and nuclear stain will
to smooth out any wrinkles caused from slicing the be lessened.
specimen. 4. Bluing (Ammonia)
• Following the water bath, the specimen is placed on a • Two dips in ammonia water
slide. Depending upon the type of staining required, the • Serves as bluing agent
slide is either stained manually or by an automated 5. Dehydration before cytoplasmic staining
stainer machine. • Use of ascending grades of alcohol
• Following staining, a protective cover slip is added to the • Prepares the cell sample for uptake of the counter
slides. stain
• Once complete, the slide is sent to a pathologist for 6. Cytoplasmic staining
examination. • Orange G-6: 5 dips; stains keratinized cell
• Eosine azure: 30 seconds; stains rest of the cell
EVERYTHING ABOUT PAPANICOLAOU STAIN
7. Clearing
• Alcohol replaces with xylene
What is the main use of Paps Stain?
• Refractive index similar to glass slides, cover slip and
• Use to stain paps smear which is used for cervical cancer mounting medium
screening in gynecology. 8. Blow dry and cover slipping (mounting)
• Most important stain (routine stain) in the practice of • Helps to protect from dust
cytopathology. • Preserve the slide
• Use to stain alcohol fixed cytology slides.
FROZEN SECTION (CRYOSTAT)
What are the stains in Papanicolaou stain?
Several things you should be aware:
• Polychromatic pap stain contains 5 dyes in 3
solutions: • Always wear gloves to avoid frostbite. (Frostbite: injury
o 1 nuclear stain (hematoxylin) caused by freezing of the skin and underlying tissues.)
o 2 cytoplasmic counter stain (OG-6 and EA-50) • Tissue freezing medium: advanced formulation that
• Nuclear stain (Hematoxylin) designed to support tissue during cryotomy. Place around
o Stains cell nuclei in BLUE the tissue or in the mold when you are preparing to go on
o Both progressive and regressive nuclear the block to be cut.
staining can be used • Pick them up with microscope slide. Use them in room
o Harris Haematoxylin: commonly used temperature for the frozen sample to adhere well.
(regressive technique) • Consult your manual for the temperature suitable for the
• 2 Cytoplasmic stain (OG-6 & EA-50) specimen.
o Orange G-6: first acidic counter stain;
monochrome stain; stains the protein keratin (a
Page 37 of 38
Parts of Cryotome
• Thickness indicator: can set to very high or very low

depending on the specimen to be cut out.
• PE button: extra freeze samples
• Clock: length of time which cooling may be maintained
• Light button: to image clearly the samples
• Specimen disc: magnetic stainless steel discs for
mounting
• Mounting area: to secure the tissue block
• Anti-roll cover: to flatten the sliced tissue
• Blade level: to lock and unlock the blade
• Stage lock: forward and backward
• One arrow button: moves the mounting area
microscopically
• Two arrow button: moves the mounting area at a greater
distance
• Handwheel: moves the mounting area on upward and
downward movements
Sample Preparation
1. Apply the media direct to the specimen.

2. Specimen should be on the center.
3. Use the heat extraction bar to immediately freeze the
tissue sample.
4. Ideal for large tissue like breast mass and thyroid organ.
Sectioning
1. Put the specimen disc in the mounting area.

2. Figure out how far away is the tissue to the blade.
3. May or may not initially see the tissue slices.
4. You can always manipulate the thickness of the slice.
5. Using the brush technique.
6. Pick up with microscope slide.
Staining
1. Immersed with hematoxylin stain for 1-2 minutes.

2. Wash well in distilled water for 10 dips.
3. 1 dip in ammonia water.
4. Rinse in distilled or running water for 10 dips.
5. Counterstain in Eosin Y for 1-2 dips.
6. Dehydrate in 2 levels of 95% ethyl alcohol.
7. Blow dry the slides.
8. Apply a protective cover slip.
9. Slide is now ready for microscopic examination.
Page 38 of 38

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analytical variable including analytical (equipment and

reagent problems) or technical errors during analysis to

• Gloves must be worn at all times HISTORY AND IMPORTANCE

• SD Urometer 720 Urine Analyzer Parameters

Components and keyboard:

2. pH (concentration of hydrogen ions)

a. Place 5mL of Benedict’s reagent into a test tube.

How is the test done?

a. Pipette 2mL of Exton’s reagent and place on the tube.

Trace Very slight cloudiness is observed

Note: this test is only one measurement of your liver function

• Nitrites detects the presence of bacteria

Note: urine-bilirubin measurement is common in urinalysis dipsticks, and

Note: dipstick test can only detect acetoacetate and acetone

8. Bilirubin and Urobilinogen

• Thick, slimy substance that coats and moistens certain

• Quantified as number of cells / HPF

• Suggests that sample was contaminated

• Refers to a series of laboratory tests done on fecal

Processing of Routine Urinalysis • Collected in a clean container (plastic or glass) with

1. With a pencil or marker, write the patient’s name or

Process of Fecal Occult Blood Test Process of Pregnancy Testing

Note: if sample collected at home, delivered to laboratory within 1 hour

1. Making a wet preparation.

𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑥106 𝑝𝑒𝑟 𝑚𝐿) 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓

𝑠𝑝𝑒𝑟𝑚𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 100,000

4.7. To get the total sperm count, multiply the sperm

𝑡𝑜𝑡𝑎𝑙 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑢𝑛𝑡 = 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒

= 13.3𝑥106 𝑝𝑒𝑟 𝑚𝐿 𝑥 2𝑚𝐿

= 26.6 𝑥106 𝑝𝑒𝑟 𝑒𝑗𝑎𝑐𝑢𝑙𝑎𝑡𝑒

5. Record and print your result.

2. Selective Media Process of Culture Media Preparation

Clinical Specimens for Aerobic Culture Upper Respiratory Tract

1. Sterile samples 1. Nasopharyngeal swab

1. Midstream clean catch

Abscess Blood (Conventional Method)

Throat Swab 3. Perform isolation streak technique.

Urine Inoculate the BAP by continuous streaking, spread the

1. Growth on BAP only

3. Growth on CAP b. Reincubate the BCB until 7 days at 36±10C,

1. Growth on BAP only

• Quantity (light / moderate / moderately heavy / heavy growth)

2. Growth on BAP and MAC

1. Growth on BAP only

2. Growth on BAP and MAC e. STY

1. Observe for a typical colony on the following culture

Culture Media Suspected Possible

2. Perform biochemical tests / serological typing on

BIOCHEMICAL TESTS: IDENTIFICATION OF

• Used to determine the ability of bacteria to utilize sodium

• Tests the metabolism of sugar by prokaryotic cells. Cells

• Used to determine if the microorganism that possesses

IDENTIFICATION OF STAPHYLOCOCCUS SPP.

• Used to differentiate Staphylococcus (+) from

• Procedure: Pick a colony from an 18–24-hour culture

Coagulase Test IDENTIFICATION OF STREPTOCOCCUS /

Optochin Susceptibility Test • Tests the ability of organisms to hydrolyze esculin in

• Tests the ability of organisms to grown in high