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Module 5 Reagents, Stains, Supplies

By Craig Baker,2014-05-07 13:51
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Module 5 Reagents, Stains, Supplies

    Module 5

     Preparation of Ziehl-Neelsen Reagents

    To provide you with an understanding of staining reagents and Purpose their preparation in acid fast staining

    Prerequisite Module 2

    Modules

    Learning At the end of this module, you will be able to Objectives ? Describe the importance of using quality chemicals for

    reagent preparation

    ? Prepare reagents required for the Ziehl-Neelsen method

    ? Describe the safety requirements for reagent preparation

    ? Use positive and negative control slides for the quality

    control of Ziehl-Neelsen reagents

    ? Explain the use and frequency of routine quality control

    procedures.

    ? Equipment required for staining reagent preparation Content Outline

    ? Reagents required for staining reagent preparation

    ? Methods for staining reagent preparation

    ? Storage of staining reagents

    ? Quality control (QC) of freshly prepared staining reagents

    Laboratory Practical Session # 1: Reagent preparation Handout and

    Exercises Laboratory Practical Session # 2: Quality control of staining

    reagents

    Appendix 1: Worksheet for preparation of reagents for Appendix

    Laboratory Practical Session # 1

    Appendix 2: Example of logbook for quality control of staining

    reagents and blank sheet

    Appendix 3: Recording worksheet for Laboratory Practical

    Session # 2

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 1 of 11

    Module 5: Preparation of Ziehl-Neelsen Reagents

Reagent preparation requires equipment for weighing and measuring. Water,

    free of environmental mycobacteria, must also be available. Environmental

    mycobacteria often colonize water tanks and taps, and could on rare occasions

    result in a false positive reading. Therefore, the intermediate-level laboratory

    usually prepares staining reagents with quality control checks before these

    staining reagents are distributed to the peripheral labs.

Good staining reagents, especially those made with a high-quality basic fuchsin

    dye, are essential to detect acid-fast bacilli (AFB). While it is easy to

    demonstrate AFB in a highly positive smear, only a good staining reagent will

    also be able to show the AFB when they are rare or damaged due to drug

    treatment and are especially difficult to stain. Poor quality staining reagents may

    not show these AFB and a case of TB maybe missed.

EQUIPMENT REQUIRED FOR STAINING REAGENT PREPARATION

     The following list is required for preparing staining reagents:

    ? A balance or weight scale, with a sensitivity of 0.1 gram

    ? Measuring cylinders of 100 mL, 500 mL, and 1000 mL capacity (one each)

    ? Large Erlenmeyer (conical) flasks or flat-bottomed balloon flasks, capacity

    at least one liter

    ? A spirit lamp for heating

    ? A stirring plate with heating and magnetic stirrers (this is preferable when

    preparing larger quantities)

    ? Containers for the newly prepared staining reagents (dark amber glass

    bottles are recommended, but plastic bottles or containers with tight

    closures may be easier to transport)

    ? Labels for bottles

    ? Brushes to clean bottles before reuse

    ? Funnels to fill bottles, one funnel for each solution

    ? AFB-positive and negative unstained control smears

REAGENTS REQUIRED FOR STAINING REAGENT PREPARATION

These will vary according to guidelines and supplies issued by your NTP. Always

    follow NTP instructions.

Carbol fuchsin

    Since many brands of basic fuchsin on the market do not specify quality, it is

    imperative to buy reputable products from branded international companies or

    any other company that has been certified by the Biological Stain Commission.

For carbol fuchsin, the following items are required:

    ? Basic fuchsin powder of good quality

    ? Phenol crystals of good quality. The crystals should be almost colorless;

    quality must be assessed through quality control of AFB staining. AFB

    smears should yield solid, homogenous, and strong red-staining bacilli.

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 2 of 11

    ? Alcohol (can be denatured 95% ethanol or methanol)

    ? Water (distilled or purified)

Decolorization solution

    For staining reagent preparations using acid, you need the following items:

    ? Concentrated sulphuric acid (> 95%)

    ? Water (distilled or purified)

    or

    ? Hydrochloric acid (37%, fuming)

    ? Alcohol (denatured 95% ethanol or methanol)

Both acids can be of industrial grade, but they should not look dirty.

Methylene blue

    For staining reagent preparations using methylene blue, you need the following

    items:

    ? Methylene blue powder of good quality

    ? Water (distilled or purified)

METHODS FOR STAINING REAGENT PREPARATION

Quantities of reagents needed may vary according to final concentrations

    prescribed by your NTP guidelines (some prefer a 0.3% fuchsin concentration,

    while others suggest 1%; some NTPs prefer 0.1% methylene blue over 0.3%).

    Always follow NTP instructions.

The quality of basic fuchsin varies from different manufacturers in its purity and

    solubility. If dye purity is known it should be taken into account for basic fuchsin,

    especially if the 0.3% final stain concentration is to be used. The amount to be

    weighed has to be corrected by dividing the prescribed amount by the decimal

    equivalent of the dye content. For instance, if a brand has a 75% dye content,

    you must divide the amounts by 0.75. So 3 g / 0.75 = 4 grams will be weighed for

    the 0.3% stain. No corrections are needed if it is 85% or higher in final

    concentration.

If the dye purity is unknown or if the basic fuchsin dissolves poorly or precipitates

    out of solution even after initial filtering of the stain, it may be prudent to use the

    higher concentration (1%) in the staining reagent preparation. This can help

    ensure that the minimal concentration of 0.3% carbol fuchsin is achieved.

Prepare the final solutions according to the following guidelines:

    ? 1 liter of carbol fuchsin: final concentrations 0.3% basic fuchsin and

    5% phenol

    ? Weigh 3 grams of basic fuchsin and 50 grams of phenol crystals

    separately

    ? Measure 100 mL of alcohol (denatured ethanol or methanol) and pour

    it in a conical flask

    ? Add the phenol and swirl the flask till it is dissolved

    ? Add the basic fuchsin powder and continue to swirl until the fuchsin

    powder completely dissolves. If this is too difficult, try adding 100 mL

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 3 of 11

    water. Check for remaining powder or crystals on the bottom. If these

    are seen, continue swirling with occasional slight heating. It may be

    preferable to leave the mixture stirring with slight heating overnight.

    ? Only after fuchsin is completely in solution, add 850 mL of water (or the

    remainder) and mix by further swirling

    ? 1 liter of sulfuric acid 25%

    ? Add 750 mL of pure (cold) water to a two liter Pyrex conical flask

    ? Measure 250 mL of concentrated sulphuric acid in a cylinder

    ? Pour it slowly into the flask containing the water, directing the flow of

    acid gently along the inner side of the flask. This will generate a lot of

    heat. Usually, it is necessary to stop a few times, swirling the flask to

    let it cool off a bit, or even holding it under the tap with cold water.

    ? Mix well by swirling the flask.

Never add water to acid! This will make it boil immediately and it may even

    splash in your face!

Always wear protective laboratory coats, gloves, and safety glasses when

    handling strong acids.

In case of an accident with acid, rinse the affected body part or cloth

    immediately with plenty of water.

    ? 1 liter of 3% hydrochloric acid in alcohol

    ? Add 970 mL of 95% alcohol to a two liter Pyrex conical flask

    ? Measure 30 mL of concentrated hydrochloric acid in a cylinder

    ? Pour it slowly into the flask containing alcohol, directing the flow of

    acid gently along the inner side of the flask with constant swirling.

    ? Mix well by swirling.

Always wear protective laboratory coats, gloves, and safety glasses when

    handling strong acids.

In case of an accident with acid, rinse the affected body part or cloth

    immediately with plenty of water.

    ? 1 liter of methylene blue 0.3%

    ? Weigh 3 grams of methylene blue powder

    ? Add the powder to 0.5 liter of pure water, which has been placed in a

    conical flask

    ? Swirl the contents of the flask to dissolve the dye

    ? Add another 0.5 liter of water and mix again

Next steps

    Let the flasks with freshly prepared reagents stand (covered) until quality control

    procedures have been performed.

    After these reagents have passed quality control, pour the solutions into clean

    bottles and label them. If bottles are reused, clean them thoroughly. Carbol

    fuchsin crystals stick to the bottom and are hard to remove, use acid alcohol and

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 4 of 11

    a bottlebrush to remove this residue. On the label of the bottle, clearly print the reagent name, concentration and the preparation date.

    Carbol fuchsin staining reagent can be filtered in the laboratory that prepared it, but this is not sufficient since it may precipitate again. Filter carbol fuchsin again during the process of staining, using a funnel with filter paper. The other staining reagents do not need to be filtered.

STORAGE OF REAGENTS

    Well-prepared reagents will keep for at least six months to one year, even at higher temperatures. Store all reagents in clean and tightly closed bottles with a label showing the name of reagent and the date of preparation. Keep these bottles out of direct sunlight. If clear bottles are used, keep stocks of reagents in a closed cabinet.

QUALITY CONTROL

PREPARATION OF POSITIVE AND NEGATIVE CONTROLS

    Make positive control smears with low positive (1+) sputum. Let this sputum stand for one or more days at room temperature to allow the sputum to liquefy. Then, with the container closed, mix the contents carefully and make as many smears as possible from this same low positive sputum. Check the average number of AFB by staining a few randomly selected smears from the entire batch. Record this number in your staining reagent logbook.

Ensure that sputum used to prepare negative control smears has been

    extensively examined to ensure that there is no AFB. Prepare smears and fix them.

    To protect fixed unstained smears from dust and sunlight, store them in a separate and labeled slide box.

QUALITY CONTROL OF FRESHLY-PREPARED STAINING REAGENTS

    When preparing staining reagents, always perform quality control for each batch of staining reagents prepared. Quality control is essential to ensure that the staining reagents work well, and that they do not contain contaminating AFB.

    It is more efficient to prepare bigger batches if very large flasks are available.

Keep accurate records in a logbook for quality control (see Appendix 2). This

    serves as an important reference record to defend against possible complaints on bad staining reagents. In the logbook, identify the batches by name of reagent and preparation date (as on the bottle labels). Perform QC by using one or more freshly prepared staining reagents and the normal staining procedure as described for positive controls. Test the performance of carbol fuchsin by staining and examining two low positive smears stained once, and two negative smears stained three times.

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 5 of 11

    Negative smears are stained three times to check for the presence of

    environment mycobacteria in the water used to prepare the reagents.

Examine all controls carefully for number, completeness, and intensity of red

    color of AFB, as well as color and complete destaining of background with

    absence of crystals. Record the results in logbook for quality control of staining

    reagents as in the example.

If unsatisfactory results are obtained in the staining of AFB, check for the method

    of preparation of carbol fuchsin and other reagents. If preparation procedure seems to have been correct, the stain might be good but the staining procedure

    not correctly used. Ensure that proper staining technique was followed. Repeat

    few more control slides, paying attention to correct staining technique. If no error

    found in the preparation method or staining technique, then prepare fresh

    staining solution(s) or reagents from a new batch of stains or reagents and

    perform quality control.

Report the unsatisfactory batch and discard the unsatisfactory solution(s).

QUALITY CONTROL OF STORED STAINING REAGENTS

Staining reagents may spoil with aging. In addition, the staining procedure may

    not have been performed correctly. For these reasons check staining periodically

    in all labs. Include a positive control smear (as described above and prepared by

    these laboratories themselves) in the routine series. Perform the QC at least

    weekly and with every new batch of reagents. Check the control smear first for

    properly stained AFB, and record the result in the sputum smear register. If the

    result is unsatisfactory, stain another control smear, making sure that the

    procedure is correct. If this gives a good result, use this lot to stain routine

    smears. If it does not, use a new lot of staining reagent to restain them. Make

    sure that the new lot has been properly quality controlled.

     Key messages ? Use quality reagents to prepare staining reagents.

    ? Accurate preparation of reagents is critical to obtain

    optimum staining results.

     ? Quality control of staining reagents using control

    smears ensures proper performance of newly prepared

    staining solutions.

    ? Record quality control results in logbook for quality

    control of staining reagents.

    ? Store prepared reagents in clean bottles and out of

    direct sunlight.

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 6 of 11

    Module Review: Module 5

Find out how much you have learned by answering these questions.

    Why must quality reagents be used to prepare staining reagents?

     _________________________________________________________________

     _________________________________________________________________

     _________________________________________________________________

Why is correct preparation of reagents necessary to obtain optimum staining

    results?

    __________________________________________________________________

    __________________________________________________________________

    __________________________________________________________________ What is the role of control smears in evaluating the performance of newly-prepared staining solutions?

    __________________________________________________________________

    __________________________________________________________________

    __________________________________________________________________

How should reagents be labelled and stored?

    __________________________________________________________________

    __________________________________________________________________

    ________________________________________________________________

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 7 of 11

Appendix 1

    Worksheet for Preparation of Reagents

Carbol fuchsin

    Item Lot number Actual amount taken

    Basic Fuchsin (in grams)

    Phenol

     (in grams)

    Alcohol (in ml)

    Water (in ml)

Counter stain

    Methylene Blue

     (in grams)

Water (in ml)

Decolorizing solution

    Sulphuric acid (in ml)

    Water

    Hydrochloric acid (in ml)

    Alcohol (in ml)

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 8 of 11

Appendix 2

    Example of Logbook for Quality Control of Staining Reagents

Batches checked on date 1/5/02:

    Carbol fuchsin (CF) batch 1/5/02, sulfuric acid batch 1/5/02, methylene blue batch

    1/5/02

     Average grading positive controls: no. 345 = 30/100 fields; no. 411 = 22/100

    fields

    Control slide AFB color AFB number Destaining Decision 345/12 strong red 20/100 F OK Accept CF 411/25 strong red 50/100 F OK Accept CF NEG NA none OK Accept others NEG NA none OK Accept others

Batches checked on date 1/15/02:

    Carbol fuchsin (CF) batch 1/15/02, sulfuric acid batch 1/15/02 (+ old methylene blue

    solution)

     Average grading positive controls: no. 345 = 30/100 fields; no. 411 = 22/100 fields

    Control Slide AFB color AFB number Destaining Decision 345/13 weak red 2/100 F OK Reject CF 411/26 NA 0/100 F OK Reject CF NEG NA none OK Accept others NEG NA none OK Accept others

Note: This batch of carbol fuchsin is bad; all has been discarded. Sulfuric solution is

    OK.

Batches checked on 1/16/02:

    Carbol fuchsin (CF) batch 1/16/02, Methylene blue batch 1/16/02 (+ old sulfuric acid solution)

     Average grading positive controls: no. 345 = 30/100 fields; no. 411 = 22/100

    fields

    Control Slide AFB color AFB number Destaining Decision 345/14 strong red 34/100 F OK Accept CF 411/27 strong red 40/100 F OK Accept CF NEG strong red 3 clumps OK Reject others NEG NA none OK Accept others

Note: contamination, probably methylene blue. To be checked further using only 1 of

    the new staining reagents on negative controls (+ old good stains of the other types).

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 9 of 11

     Blank Log Book Sheet:

     Quality Control of Staining Reagents

    (This sheet can be reproduced for individual laboratory use)

Batches checked on date :_______

    Carbol fuchsin (CF) batch ______, sulfuric acid batch _______, methylene blue batch _______

    Average grading positive controls: no. ____ = ____/100 fields;

     no.____= _____/100 fields

    Control slide AFB color AFB number Destaining Decision

Note:

Batches checked on date:______________

    Carbol fuchsin (CF) batch ________ sulfuric acid batch _______ methylene blue Batch________

    Average grading positive controls: no. ____ = ____/100 fields;

     no. ___ = ____/100 fields

    Control Slide AFB color AFB number Destaining Decision

Note:

Batches checked on ________

    Carbol fuchsin (CF) batch _________, Sulfuric acid solution________, Methylene blue batch _______

    Average grading positive controls: no. _____ = ____/100 fields; no. _____ = _____/100 fields

    Control Slide AFB color AFB number Destaining Decision

Note:

     Module 5: Preparation of Ziehl-Neelsen Reagents Page 10 of 11

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