Direct Microscopic Flashcards
What does the direct microscopic method allow analysts to examine in milk?
Numbers of bacterial clumps or somatic cells
This method is also known as the direct microscopic clump (DMC) count.
What can be evaluated simultaneously with the counting of bacterial clumps?
Morphology and arrangement of bacteria
This evaluation helps identify quality problems.
What do large numbers of bacterial clumps indicate?
Mastitis or other abnormal conditions of the udder
This is a significant indicator of udder health.
What term has come to replace ‘leucocytes’ in the context of somatic cells?
Somatic cells
This change reflects a more meaningful expression of body cells associated with the udder.
What is the official term for the microscopic method that counts somatic cells?
Direct microscopic somatic cell count (DMSCC)
This method is recognized for its research findings and application.
How were somatic cell counts previously estimated before the DMSCC?
By one of several other testing methods
These previous methods were less direct than the current microscopic approach.
What is the primary limitation of the direct microscopic method in pasteurized milk?
It is not used to determine compliance with finished standards
This is due to the generally low numbers of bacteria in pasteurized milk.
Why might the direct microscopic method still be useful in quality control?
Large numbers of bacteria in uncultured milk indicate unsanitary conditions
This applies regardless of whether the bacteria are viable.
Fill in the blank: The direct microscopic clump count is used to assess the _______ of milk.
Quality
This assessment can help identify potential health issues in dairy cows.
What does DMC stand for in the context of milk analysis?
Direct Microscopic Count
DMC is used to evaluate the bacterial contamination of raw milk or cream samples.
What is the primary purpose of evaluating the morphology of cells in milk samples?
To suggest the causes of high bacterial counts
This evaluation includes assessing the configuration of clumps and the presence of somatic cells.
True or False: Most dry milk contains a high number of viable bacteria.
False
Most dry milk contains relatively few viable bacteria.
What can microscopic examination of reconstituted dry milk samples provide?
Meaningful information on the previous sanitary history of the product
This examination can serve as a quality control measure.
What is the relative standard deviation (RSDR) in counts made on the same sample of milk?
3.7
This indicates potential for wide interlaboratory differences.
Fill in the blank: The number of somatic cells in raw milk provides a measure of the presence and extent of _______.
mastitis
Somatic cell counts help assess one aspect of raw milk quality.
What are the two types of counts mentioned in the context of milk analysis?
- Direct Microscopic Counts
- Direct Microscopic Somatic Cell Counts
These counts measure bacteria and somatic cells per milliliter or gram of dairy product.
What is the principle behind direct microscopic counts?
Bacteria or somatic cells are transferred to a slide, stained, and counted
This yields the number of cells per milliliter or gram of dairy product.
What type of immersion oil is preferred for microscopy in this context?
Type B (medium viscosity)
The refractive index should be 1.51 to 1.52 at 20°C.
What is the significance of using clear glass slides with etched surfaces?
They allow for confirmation of the correct reading area during analysis
This is important for achieving high accuracy.
How should slides be cleaned for repeated use?
Submerge in hot detergent solution and rinse thoroughly
Alternatively, use appropriate commercial alkaline and/or acid cleaners.
What instrument is mentioned for transferring 0.01-mL quantities?
Metal syringe
This instrument allows for rapid and convenient transfer but can create artifacts in milk films.
What should be done immediately after using a metal syringe?
Disassemble and clean the measuring tube and piston
This prevents residues from drying on the instrument.
What should be avoided when cleaning the syringe barrel and finger grip?
Do not submerge in detergents or solvents.
Proper maintenance prevents contamination and artifacts.
How should the interior of a syringe be cleaned?
Use forced suction or pressure with a rubber bulb and soapless detergent, then rinse with clean water and air-dry.
Acids, alkalis, and ordinary soap solutions should not be used.
What pH level should chemical cleaners be at when used for syringes?
At or near pH 7.0.
This ensures compatibility and safety for the syringe materials.
What is the first step before reassembling a syringe?
Ensure all detergent and milk residues are removed.
Proper cleaning is crucial for accurate results.
What precaution should be taken regarding syringe parts?
Do not interchange parts from one syringe with parts from another.
This prevents compatibility issues and maintains accuracy.
How often should syringes be tested for accuracy?
Annually.
Certification may be required by regulatory agencies.
What should be done if a syringe does not conform to specified milk deliveries?
Replace the syringe.
Ensures reliability in measurements.
How is the calibration of the volumetric load of a syringe determined?
By weight measurements of a representative test charge of milk.
This method ensures precision in delivery.
What is the average weight difference expected in a correctly calibrated syringe after multiple weighings?
0.0103 + 0.0005 g.
This value indicates proper calibration.
What are micro pipeters used for?
Measuring the 0.01-mL milk test portion.
They are preferred instruments due to their precision.
What is the purpose of a bent-point needle in milk testing?
Used for spreading milk or cream over a square centimeter area.
This facilitates even distribution for analysis.
What temperature range is recommended for drying films?
40° to 45°C.
Uniform heat distribution is important for consistent results.
What type of microscope is preferred for milk analysis?
A binocular microscope with a 1.8-mm oil-immersion objective.
Additional features include an Abbe condenser and a numerical aperture of 1.25 or higher.
What is the recommended magnification for oculars in milk testing microscopes?
10x magnification is recommended, with 12.5x to 15x permitted.
This ensures sufficient detail for analysis.
What is the significance of the microscopic factor (MF) in milk analysis?
It determines the amount of milk that can be examined in any one field.
MFs are used to calculate bacterial clumps or somatic cells per field.
What should be ensured about the surface for drying films?
It should be clean, dust-free, and level.
A regulated heat source is also necessary.
Fill in the blank: Syringes should be cleaned before _______.
first use.
True or False: It is acceptable to use ordinary soap solutions for cleaning syringes.
False.
Ordinary soap solutions can damage the syringe.
What should be adjusted for individuals using paired oculars in microscopy?
Diaphragms must be specially adjusted for the individual
Counts must be corrected based on the diameter of the field observed.
What is the significance of constant tube length in microscopes?
It keeps the resultant calibrated field and magnification constant regardless of changes in interpupillary settings.
What is the MF in microscopy?
Magnification Factor
It is calculated based on the field diameter observed.
How is the Magnification Factor (MF) calculated?
MF = 3.1416 x d² / 40,000
‘d’ is the diameter measured under the oil-immersion objective.
What is the formula to express the MF?
Express the MF to 2 significant figures.
What does the reciprocal of the MF represent?
The fraction of 1 mL of milk observed in 1 microscopic field.
What is the purpose of the ocular disc in microscopy?
To subdivide the field for counting purposes.
What type of stage is required for microscopy?
A regular mechanical stage or a special stage for specific slide sizes.
What is a stage micrometer slide used for?
To measure field diameter accurately.
What is a hand tally used for in microscopy?
To count cells or particles observed.
At what temperature should samples be warmed before testing for DMSCC?
40°C.
How should samples be mixed before preparing films?
Shake 25 times in 7 seconds with a 1-foot movement.
What is the maximum time to allow foam to disperse after mixing?
No more than 3 minutes.
What is essential for cleanliness when preparing films?
Instruments and glassware must be scrupulously clean.
What is the drying temperature range for films?
40° to 45°C.
What should be done with the measuring tube before each use?
Rinse it in fresh tap water.
What is the correct technique for using a 0.01-mL syringe?
Dip the tip slightly below the surface and repeatedly draw in and expel water.
What should be avoided when wiping the tip of the syringe?
Do not wipe over the tip.
What is the procedure for using micro pipeters?
Use a separate tip for each sample and avoid foam.
What should be done with used tips from micro pipeters?
Discharge into disinfectant or biohazard bags.
What is a key consideration during film preparation to prevent contamination?
Protect films from exposure to dirt, dust, and insects.
What should be done with a clean bent-point dissecting needle between samples?
Wipe the needle on a clean, dry tissue or towel
At what temperature should the milk film be dried?
40° to 45°C
What is the recommended method for handling multiple slides during staining?
Use holders of glass, stainless steel, or other suitable material
What should be avoided when preparing stains?
Using liquid reagents that contain suspended foreign matter
Which three stains may be used for cow’s milk?
- LW/NL-T stain
- LW/NL-X stain
- LW/NL-F stain
What is the issue with the LW/NL-T stain when used for goat’s milk?
It has produced highly variable results and is unreliable for somatic cell count
What is a suitable replacement for staining somatic cells in goat’s milk?
Pyronin Y methyl green stain with Carnoy’s fixative
What are the components for the LW/NL-T stain?
- 0.6 g of certified methylene blue chloride
- 52 mL of 95% ethyl alcohol
- 44 mL of tetrachlorethane
- 4 mL of glacial acetic acid
How long should the LW/NL-T stain solution stand?
12 to 24 hours at 44° to 72°C
What is the difference in the preparation of LW/NL-X stain compared to LW/NL-T stain?
LW/NL-X uses xylene instead of tetrachlorethane and 0.5 g of methylene blue chloride
What is included in the Canadian modification of the modified Newman-Lampert stain?
Basic fuchsin dye
What are the components for Carnoy’s fixative?
- 60 mL of chloroform
- 20 mL of glacial acetic acid
- 120 mL of 100% ethyl alcohol
What should be done with the pyronin Y stain before use?
Filter through Whatman No. 1 paper
How should dried, stained slides be rinsed after staining?
In 3 changes of tap water at 35° to 45°C
What is the requirement for the New York modification of the pyronin Y-methyl green stain procedure?
Use of a fume hood
How long should a prepared film be fixed in Carnoy’s fixative?
5 minutes
What should be done with used staining solutions?
Discard them whenever they become contaminated or unsuitable
How should slides be stored after examination?
In dust-free, slide-storage boxes
What can interfere with the staining procedure or future re-examination of milk films?
Accumulation of pencil marks on slides
What is the criteria for counting bacteria in separate clumps?
Count as separate clumps if 2 cells or clumps are separated by a distance equal to or greater than twice the smallest diameter of the 2 cells nearest each other.
What cells are counted in DMSCCs?
Only somatic cells with an identifiable stained nucleus.
What color is the stained nucleus in bovine somatic cells?
Dark blue.
What color is the stained nucleus in caprine somatic cells?
Blue or blue-green.
How do you count polymorphonucleated cells?
Count as a single cell if it has two or more discernible nuclear lobes.
What is the minimum diameter for a nucleus to be counted?
8 microns.
What is the formula to calculate the area of a single strip in the counting method?
Area of a single strip (mm²) = 11.28 mm x D (in mm).
How do you determine the number of single strips in a 0.01-mL milk film?
Number of single strips = 100 mm² / area of single strip.
What is the Single Strip Factor (SSF)?
SSF = Number of strips in 0.01 mL x 100.
What is the counting procedure for a single-strip count?
Focus on the film edge, traverse the diameter, counting cells within the strip and those touching one edge.
How is the count per milliliter calculated?
Count per milliliter = Number of somatic cells and/or bacterial clumps in a single strip x SSF.
What is the importance of analyst training in counting accuracy?
The training and skill of the analysts are crucial for accuracy, precision, and reproducibility.
What are some factors responsible for variations in counts?
- Inaccuracy in measuring samples * Faulty preparation and staining * Minute amount of milk examined * Irregular distribution of cells * Poor microscopy * Eye fatigue * Analyst inexperience.
How should counts be reported?
Report counts only to the first 2 left-hand digits of the estimate, followed by the appropriate number of zeroes.
What is the rule for rounding when the third digit is 5?
If the second digit is odd, round up; if even, round down.
What should be done with slides that have violative counts?
Save slides for future reference.
