MODULE: LC1

Culture techniques

1. Aseptic Culture techniques
1. Use Universal Precautions for protection against potential exposure to infectious agents (e.g., protective clothing, gloves and masks, containers for sample delivery and waste disposal, biological safety cabinets).
2. Use and document methods to detect, identify, control, and eliminate microbial or chemical contamination.
3. Practice measures that prevent cross-contamination between samples.
2. Media Preparation Techniques
1. Choose appropriate medium additives such as sera, antibiotics, buffers, mitogens, and growth factors depending on sample type and test ordered.
2. Select appropriate methods of preparation and storage of media to maintain pH, sterility, and ability to support growth. Document processes to exclude expired reagents.
1. Appropriate culture techniques for specimens
1. Select culture equipment and vessels for closed or open culture systems.
2. Select culture technique for specimen taking into account type of tissue, methods of initiation, type of culture, and purpose for study.
3. Appraise the effect of cell density on rate of growth and adjust appropriately (e.g., cell count of leukemic specimens).
4. Monitor and document the effectiveness of all solutions used in the procedures prior to use on diagnostic material.
5. Record complete information for culture of specimen, including identification of technologist, lot numbers of media, sera, growth factors, and other reagents, incubator used, and mitogen, if used
2. Monitor cell growth and control variables.
1. Employ measures that will maintain optimal cell growth (e.g., feeding and centrifugation/concentration of specimens prior to culture to correct for depleted medium).
2. Evaluate status of cultures using assessment of growth and mitotic activity, pH of medium, and turbidity.
3. Identify and document probable causes of poor growth and culture failure, such as inadequate specimens, or equipment failure, and describe corrective actions taken.
4. Report findings of culture failure or growth inadequate for analysis to laboratory personnel, and, under supervision as needed, request new sample, if appropriate.
3. MODULE: LC2

Principles and Techniques for Harvesting cell cultures

1. Determine optimal time sequence and method for harvest (manual or robotic)

Apply knowledge of cell cycle for various cell types and culture conditions (e.g., PHA stimulated lymphocytes, unstimulated leukemic cells, synchronized cultures) to time harvests

4. Harvesting procedures for specimen or culture
1. Understand the use synchronizing or intercalating agents, such as amethopterin, fluorodeoxyuridine, bromine deoxyuridine, ethidium bromide, or echinomycin D, at appropriate concentration, temperature, and duration.
2. Use spindle fiber inhibitor (e.g., Colcemid, Velban) at correct concentration, temperature, and duration.
3. Use recommended procedure for removing cells from culture vessels. Use appropriate hypotonic solution (KCl or sodium citrate), at correct concentration, temperature, and duration.
4. Use cell fixative (acetic acid/methanol) at correct concentration, temperature, and duration.
5. Control mechanical damage to chromosomes by proper mixing, shaking, pipetting, centrifuging, or other handling of the cells.
6. Record complete information for harvest of specimen including date, addition of spindle fiber inhibitor, intercalating or synchronizing agents, conditions used for harvest, and name of technologist processing the samples.
5. Slide Preparation Technique for analyzable metaphases
1. Select method of slide preparation that will produce high quality metaphases with optimum spreading (e.g., control variables such as wet or dry slides, air flow, humidity level and temperature to regulate slide drying rate).
2. Employ techniques that control concentration and distribution of cellular and other debris on slides.
3. Evaluate quality of slides with phase contrast microscope and adjust variables as necessary. Describe, document processes for determining acceptability for analysis.
4. Employ techniques that control the aging of slides to produce optimal banding conditions (e.g., storing at various temperatures, such as 37°C, 60°C, 90°C, for various times, such as 20 min to 2 hours, or overnight; UV exposure or microwave).
5. Use slide storage methods that best maintain chromosome quality for banding and staining procedures, with protection from humidity, light, chemicals, or mechanical damage
6. MODULE: LC3
1. Principles and techniques of Chromosome Banding & Staining
7. MODULE: LC4
1. Maintenance and use of microscopes and Software’s/ computer-generated imaging techniques and equipment
8. MODULE: LC5
1. Chromosome analysis (KARYOTYPING),
9. MODULE: LC6
1. FISH Analysis (REPORTING)
 

PROFICIENCY TEST:

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After successful completion of each module a presentation will be given by trainee followed by quiz for successful completion of the modules. All the Successful candidate will be given a Certificate for completion of training/dissertation work.