+-------------------+--------------------------------------------------+
| ![](media/image1. | Module 5                                         |
| jpeg){width="1.50 |                                                  |
| 83333333333333in" | **Preparation of Ziehl-Neelsen Reagents**        |
| height="1.135     |                                                  |
| 4166666666667in"} |                                                  |
+-------------------+--------------------------------------------------+

+-----------------+----------------------------------------------------+
|                 |                                                    |
+-----------------+----------------------------------------------------+
| **Purpose**     | To provide you with an understanding of staining   |
|                 | reagents and their preparation in acid fast        |
|                 | staining                                           |
+-----------------+----------------------------------------------------+
| *               | Module 2                                           |
| *Prerequisite** |                                                    |
|                 |                                                    |
| **Modules**     |                                                    |
+-----------------+----------------------------------------------------+
| **Learning      | At the end of this module, you will be able to     |
| Objectives**    |                                                    |
|                 | -   Describe the importance of using quality       |
|                 |     chemicals for reagent preparation              |
|                 |                                                    |
|                 | ```{=html}                                         |
|                 | <!-- -->                                           |
|                 | ```                                                |
|                 | -   Prepare reagents required for the              |
|                 |     Ziehl-Neelsen method                           |
|                 |                                                    |
|                 | ```{=html}                                         |
|                 | <!-- -->                                           |
|                 | ```                                                |
|                 | -   Describe the safety requirements for reagent   |
|                 |     preparation                                    |
|                 |                                                    |
|                 | -   Use positive and negative control slides for   |
|                 |     the quality control of Ziehl-Neelsen reagents  |
|                 |                                                    |
|                 | ```{=html}                                         |
|                 | <!-- -->                                           |
|                 | ```                                                |
|                 | -   Explain the use and frequency of routine       |
|                 |     quality control procedures.                    |
+-----------------+----------------------------------------------------+
| **Content       | -   Equipment required for staining reagent        |
| Outline**       |     preparation                                    |
|                 |                                                    |
|                 | -   Reagents required for staining reagent         |
|                 |     preparation                                    |
|                 |                                                    |
|                 | -   Methods for staining reagent preparation       |
|                 |                                                    |
|                 | -   Storage of staining reagents                   |
|                 |                                                    |
|                 | -   Quality control (QC) of freshly prepared       |
|                 |     staining reagents                              |
+-----------------+----------------------------------------------------+
| **Handout and   | Laboratory Practical Session \# 1: Reagent         |
| Exercises**     | preparation                                        |
|                 |                                                    |
|                 | Laboratory Practical Session \# 2: Quality control |
|                 | of staining reagents                               |
+-----------------+----------------------------------------------------+
| **Appendix**    | Appendix 1: Worksheet for preparation of reagents  |
|                 | for 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

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

```{=html}
<!-- -->
```
-   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.

-   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 ([\>]{.underline} 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

    ```{=html}
    <!-- -->
    ```
    -   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 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

    ```{=html}
    <!-- -->
    ```
    -   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**

```{=html}
<!-- -->
```
-   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%**

```{=html}
<!-- -->
```
-   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 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.

> ***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           | -   Use quality reagents to prepare staining       |
| messages**      |     reagents.                                      |
|                 |                                                    |
| ![](m           | -   Accurate preparation of reagents is critical   |
| edia/image2.wmf |     to obtain optimum staining results.            |
| ){width="1.1701 |                                                    |
| 388888888888in" | -   Quality control of staining reagents using     |
| height="0.83194 |     control smears ensures proper performance of   |
| 44444444445in"} |     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.                        |
+-----------------+----------------------------------------------------+

+-----------------+----------------------------------------------------+
| > ![](m         | Module Review: Module 5                            |
| edia/image3.wmf |                                                    |
| ){width="1.0in" |                                                    |
| >               |                                                    |
| height="0.96041 |                                                    |
| 66666666667in"} |                                                    |
+=================+====================================================+
| > 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?                         |
| >                                                                     |
| > \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\                     |
| _\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ |
| \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\ |
| _\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ |
| \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\ |
| _\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ |
+-----------------------------------------------------------------------+

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)**      |                     |                            |
+------------------+---------------------+----------------------------+

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     | AFB color   | AFB number  | Destaining  | Decision    |
| slide       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| 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     | AFB color   | AFB number  | Destaining  | Decision    |
| Slide       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| 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     | AFB color   | AFB number  | Destaining  | Decision    |
| Slide       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| 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:       |             |             |             |             |
| con         |             |             |             |             |
| tamination, |             |             |             |             |
| 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).     |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+

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;     |             |             |             |             |
|             |             |             |             |             |
| n           |             |             |             |             |
| o.\_\_\_\_= |             |             |             |             |
| \_\         |             |             |             |             |
| _\_\_\_/100 |             |             |             |             |
| fields      |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| Control     | AFB color   | AFB number  | Destaining  | Decision    |
| slide       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| 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     | AFB color   | AFB number  | Destaining  | Decision    |
| Slide       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| Note:       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| Batches     |             |             |             |             |
| checked on  |             |             |             |             |
| \_\_\       |             |             |             |             |
| _\_\_\_\_\_ |             |             |             |             |
|             |             |             |             |             |
| Carbol      |             |             |             |             |
| fuchsin     |             |             |             |             |
| (CF) batch  |             |             |             |             |
| \_\_\_\_    |             |             |             |             |
| \_\_\_\_\_, |             |             |             |             |
| Sulfuric    |             |             |             |             |
| acid        |             |             |             |             |
| sol         |             |             |             |             |
| ution\_\_\_ |             |             |             |             |
| \_\_\_\_\_, |             |             |             |             |
| Methylene   |             |             |             |             |
| blue batch  |             |             |             |             |
| \_\         |             |             |             |             |
| _\_\_\_\_\_ |             |             |             |             |
|             |             |             |             |             |
| Average     |             |             |             |             |
| grading     |             |             |             |             |
| positive    |             |             |             |             |
| controls:   |             |             |             |             |
| no.         |             |             |             |             |
| \_\_\_\_\_  |             |             |             |             |
| =           |             |             |             |             |
| \           |             |             |             |             |
| _\_\_\_/100 |             |             |             |             |
| fields;     |             |             |             |             |
|             |             |             |             |             |
| no.         |             |             |             |             |
| \_\_\_\_\_  |             |             |             |             |
| =           |             |             |             |             |
| \_\         |             |             |             |             |
| _\_\_\_/100 |             |             |             |             |
| fields      |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| Control     | AFB color   | AFB number  | Destaining  | Decision    |
| Slide       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| Note:       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+

Appendix 3

**Recording Worksheet for**

**Laboratory Practical Session \# 2**

+-------------+-------------+-------------+-------------+-------------+
| Batches     |             |             |             |             |
| checked on: |             |             |             |             |
|             |             |             |             |             |
| Carbol      |             |             |             |             |
| fuchsin,    |             |             |             |             |
| date        |             |             |             |             |
| prepared    |             |             |             |             |
| .........   |             |             |             |             |
| Con         |             |             |             |             |
| centration. |             |             |             |             |
|             |             |             |             |             |
| D           |             |             |             |             |
| ecolorizer, |             |             |             |             |
| date        |             |             |             |             |
| prepared.   |             |             |             |             |
| Type        |             |             |             |             |
| ..........  |             |             |             |             |
| ........... |             |             |             |             |
|             |             |             |             |             |
| Methylene   |             |             |             |             |
| blue, date  |             |             |             |             |
| prepared    |             |             |             |             |
| .           |             |             |             |             |
| ........... |             |             |             |             |
| Con         |             |             |             |             |
| centration. |             |             |             |             |
|             |             |             |             |             |
| Average     |             |             |             |             |
| grading     |             |             |             |             |
| positive    |             |             |             |             |
| controls =  |             |             |             |             |
| ....        |             |             |             |             |
| ........AFB |             |             |             |             |
| / 100       |             |             |             |             |
| fields      |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| **Control   | **AFB       | **AFB       | **B         | **Remarks** |
| slide ID**  | color**     | number**    | ackground** |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
|             |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+