Direct Microscopic Somatic Cell Count Flashcards
What is the maximum time unpreserved samples can be tested after initial collection?
72 hours
What are the preservatives that allow samples to be tested up to 7 days after collection?
0.02% 2-bromo-2-nitropropane-1,3-dio (Bronopol™) or 0.05% potassium dichromate (K2Cr2O7)
What is the required face velocity for a functional fume hood?
100 ft/min
What are the dimensions of the clean microscope slides used in the procedure?
2.54 x 7.62 cm
What is the diameter of the areas delineated on the microscope slides?
11.28 mm
What is the purpose of the metal syringe in the process?
For rapid and convenient transfer of 0.01 mL of milk
What is the acceptable weight delivery range for the metal syringe?
0.0103 ± 0.0005 g
What must be done annually for the fume hood?
Check, maintain records, and tag unit
What identification is required for the metal syringe?
Etched identification or imprinted serial number
Fill in the blank: Samples may be run up to _____ after initial collection if preserved.
7 days
True or False: Micropipettors are used for transferring more than 0.01 mL of milk.
False
What is the average weight to be checked for accuracy with the metal syringe?
0.0103 g
What is the significance of the accuracy check date for the metal syringe?
To ensure proper functioning and reliability
What is the average weight specified for accuracy check in item 6.e.4?
0.0103 +0.0005g
This is based on the average of 10 consecutive weighings with milk.
What should be done if using Artel PCS according to CP item 6.e.5?
See CP item 6.e.5
Specific instructions for using Artel PCS are outlined in that section.
What is required for the micropipettor used in the procedure?
Etched with identification (imprinted serial number acceptable); tag with accuracy check date
This ensures traceability and compliance with accuracy checks.
What type of needle is suitable for spreading milk film?
Dissecting Needle, Bent Point
This needle design facilitates even spreading of the film.
What temperature range should the drying device maintain?
40-45°C
This temperature range is crucial for effective drying.
What is required to monitor the temperature of the drying device?
Temperature measuring device
Accurate temperature monitoring ensures proper drying conditions.
What tools are required for dipping and holding slides?
Forceps or Slide Holder
These tools help in handling slides without contamination.
What should staining jars or trays have?
Tight fitting covers
This helps to prevent evaporation and contamination of solvents and stains.
What is the recommended storage condition for slides?
Clean, dust-free insect-proof boxes, cases or files
Proper storage conditions are necessary to maintain slide integrity.
What type of microscope is specified for use?
Binocular with 1.8 mm oil immersion objective, rack and pinion sub-stage, condenser with iris diaphragm
This microscope configuration is essential for detailed observation.
What ocular magnification is recommended for the microscope?
10X (12X or 12.5X), Huygenian or wide-field
These magnifications provide a suitable field of view for analysis.
What is the maximum Single Strip Factor (SSF) specified for the optics?
6070 or smaller
This ensures sufficient resolution for the analysis performed.
How often should each analyst measure field diameter and calculate SSF?
Annually
This regular check helps maintain accuracy in measurements.
To how many significant figures should the SSF be rounded?
Three significant figures
This precision is important for consistency in reporting.
What is the formula to compute the Single Strip Factor (SSF)?
SSF = 10,000/(11.28 x D)
D is the field diameter in mm measured using a stage micrometer.
What is the field diameter (D) used for in the calculation of SSF?
D is measured in mm using a stage micrometer.
What are the characteristics of a suitable mechanical stage for microscope examination?
Smooth action, does not drift, allows proper tracking of smears.
What is the purpose of the microscope lamp?
Provides adequate illumination.
What divisions are ruled on a stage micrometer?
0.1 mm and 0.01 mm divisions.
What is the refractive index of immersion oil?
1.51-1.52.
What is the first step in preparing the Levowitz-Weber Modification of the Newman-Lampert Stain?
Slowly add 0.6 g certified methylene blue chloride to 52 mL of 95% ethyl alcohol and 44 mL of tetrachloroethane in a 200 mL flask.
What safety precautions should be taken when making the stain?
Use gloves and prepare in a fume hood.
How long should the stain stand at the specified temperature?
12-24 hours at 4.5-7.5°C.
What type of filter paper should be used to filter the stain?
Whatman No. 42 filter paper or equivalent.
What should be added to the stain after filtering?
4 mL of glacial acetic acid.
How should the prepared stain be stored?
In a clean, tightly closed container.
What may cause problems with the stain when stored?
Traces of water or solvent.
What are alternative forms of the stain that can be used?
Commercially prepared (xylene or tetrachloroethane).
What information is required when using commercially prepared stains?
Brand, Lot #, Exp. Date.
What is Canadian Formula Stain?
Commercially prepared stain using xylene or tetrachloroethane
Includes lot number, expiration date, and brand information.
What are the components for preparing Alternate Methylene Blue Stain?
Combine:
* Cert. Methylene Blue Chloride (0.5 g)
* 95% Ethyl Alcohol (56 mL)
* Xylene (40 mL)
* Glacial Acetic Acid (4 mL)
Follow the preparation method outlined in item 14.
What is the fixative used for Pyronin Y-Methyl Green Stain for Goat, Sheep or Camel Milk?
Carnoy’s fixative
Components include chloroform, glacial acetic acid, and 100% ethyl alcohol.
What are the components for Carnoy’s fixative?
Combine:
* Chloroform (60 mL)
* Glacial Acetic Acid (20 mL)
* 100% Ethyl Alcohol (120 mL)
Can be prepared or commercially obtained.
What is the preparation method for Pyronin Y-Methyl Green Stain?
Combine:
* Pyronin Y (1.0 g)
* Methyl Green (0.56 g)
* Water (196 mL)
Filter through Whatman No. 1 paper before use.
How should Pyronin Y-Methyl Green Stain be stored?
Stain is light sensitive; store in brown bottle
Can also be commercially prepared.
What are the steps for cleaning slides?
- Physically clean
- Soak new slides in strong cleaning solution
- Rinse thoroughly in flowing water (10-15 sec)
- Soak used slides in hot detergent or wetting agent until residues are removed; rinse as above
Ensures slides are free from contaminants.
What is the recommended method for drying slides?
Air or heat dry with minimal exposure to dust, insects, etc. and store dry
What should be done to slides before use if stored in alcohol?
Flame just before use
How should each sample area on a slide be identified?
Legibly and indelibly identify each sample area on margin of slide
What is the procedure for sample agitation?
Mix samples or subsamples by shaking 25 times in 7 sec with a 1 ft movement or vortex for 10 sec at maximum setting
What is the time limit for using samples after agitation?
Use within 3 min
What temperature can high fat samples be warmed to before testing?
40°C for no longer than 10 min
What is the rinsing procedure for a metal syringe before use?
Rinse syringe 2-3 times in clean, 25-35°C tap water
How deep should the syringe be inserted into the milk before transferring the test portion?
Not over 1 cm below surface of milk
What should be done after rinsing the syringe with milk?
Fully depress and release plunger and withdraw test portion
How should excess milk be removed from the syringe tip?
With clean paper tissue, wipe downward away from tip
What is the correct technique for expelling the test portion onto the slide?
Touch the slide with the tip and expel the test portion
When spreading the milk on the slide, what should be avoided?
Do not release plunger until after touching off and removing tip from slide
What technique should be used to spread the milk on the slide?
Spread milk with point of bent needle point
What should be done between preparing multiple smears?
Complete steps 21.a through 21.e.4 before starting the next smear
At what temperature should smears be dried?
40-45°C within 5 min on level surface
What should be avoided to prevent smears from cracking and peeling during staining?
Heating too rapidly
Rapid heating can cause damage to the smear, affecting the quality of the staining process.
How should smears and slides be treated until read?
Protected from damage
Proper handling is essential to maintain the integrity of the samples.
What should be done to prevent residues from drying on a metal syringe?
Clean immediately after use
Prompt cleaning helps maintain the functionality and hygiene of the instrument.
What should not be removed from the syringe unless necessary?
Spring
The spring mechanism is critical for the operation of the syringe.
What type of detergents should be used for cleaning syringes?
Soap-less detergents and/or fat solvents
These cleaning agents are effective without leaving harmful residues.
How should residues be cleaned from the measuring tube of a syringe?
By circulating detergent with bulb on delivery end
This method ensures thorough cleaning of the measuring tube.
What should be used to clean the piston of a syringe?
Dry paper tissue
Using dry tissue prevents moisture accumulation that could affect performance.
What should be done with the micropipettor tip for each sample?
Use a new tip
This practice prevents cross-contamination between samples.
What is the correct method for withdrawing a sample with a micropipettor?
Depress plunger, insert tip below surface, release plunger slowly
This technique minimizes the introduction of foam and bubbles.
How should excess liquid be removed from the exterior of a micropipettor tip?
Wipe away from the tip with clean paper tissue
This prevents contamination of the sample being processed.
Where should the micropipettor tip be placed when preparing a smear?
Near the center of the area for smear
Proper placement ensures an even distribution of the sample.
What should be done if the pipettor has two stops when expelling sample?
Depress plunger to second stop, touch off
This ensures complete expulsion of the sample.
What technique should be used to spread milk on the slide?
With the point of a bent needle, not hockey stick style
This technique allows for a more controlled and even smear.
What should be done between samples when using a needle for spreading?
Wipe needle dry on tissue
This prevents contamination of subsequent samples.
What is the recommended drying temperature for smears?
40-45°C
This temperature range ensures proper drying without damaging the smear.
Within what time frame should smears be dried?
Within 5 minutes
Timely drying helps preserve the quality of the smear for analysis.
What should be avoided to prevent smears from cracking and peeling during staining?
Do not heat too rapidly
How should smears and slides be protected until they are read?
Protect from damage
What is the first step in the staining process using Levowitz-Weber and Methylene Blue Stains?
Use ventilated hood for steps 24.a.2-4
For how long should slides be submerged or flooded in stain during Levowitz-Weber and Methylene Blue Stains?
2 min
What should be done to drain excess stain from the slide?
Rest edge of slide on absorbent paper
What temperature should the tap water be for rinsing stained slides?
35-45°C
What is the key characteristic of the Pyronin Y-Methyl Green Stain?
Stain is light sensitive
List the steps in the staining scheme for Pyronin Y-Methyl Green Stain.
- Carnoy’s Fixative
- 50% Ethanol
- 30% Ethanol
- DI or MS Water
- Stain
- N-Butyl Alcohol
- Xylene
What is the duration for the Carnoy’s Fixative step in the Pyronin Y-Methyl Green Stain?
5 min
What color do cells stain in the Pyronin Y-Methyl Green Stain?
Blue or blue-green
What color do RNA and the background stain in the Pyronin Y-Methyl Green Stain?
Pink
What should be done if smears will not adhere to slides after Carnoy’s fixative?
Allow slide to dry for approx. 10 min, protected from light
What adjustment should be made to the microscope lamp during examination?
Adjust to provide maximal optical resolution
What is the first step to locate the edge of the smear to be read?
Use low power
What should be placed on the smear for examination?
1 drop immersion oil
What is the first step when using an oil immersion lens?
Carefully lower oil immersion lens
What should be focused on and located at the beginning of cell counting?
Center of edge of area
How should cells be counted in a smear?
Count all cells in field wide strip across diameter of a single smear
What type of cells possess a nucleus that stains dark blue in bovine secretory systems?
Somatic cells
In which secretory systems do nuclei stain blue or blue-green?
Apocrine (caprine) secretory systems
When counting cells, which cells should be included?
Cells touching one edge of the strip but not the other edge
How should fragments be counted?
Only if more than 50% of the nuclear material is visible
How should clusters of cells be counted?
Count as one unless nuclear units are clearly separated
What should be done if there is doubt about counting a cell?
Do not count
What should be recorded after examining each smear?
Record strip count
How often should comparative counting between analysts be conducted?
Monthly
What is the first step in reporting somatic cell counts?
Record of strip count for each smear examined
How is DMSCC/mL computed?
Multiply number of cells counted (strip count) by the SSF
What should somatic cell counts be reported as?
DMSCC/mL
How should the first two left-hand digits of DMSCC/mL be recorded?
Round as necessary
What rounding rule applies when the third digit is 5 and the second digit is odd?
Round up
What rounding rule applies when the third digit is 5 and the second digit is even?
Round down
What form is maintained for Direct Microscopic Somatic Cell Count?
FORM NCIMS 2400d
