• Part I
• Part II
    

This two-part, sound-slide program is on the use of fluorochrome-staining for detecting acid-fast mycobacteria. The program is intended as a guide for laboratory workers with basic to intermediate levels of mycobacteriology experience who will stain and examine smears for acid fast bacilli. Each time you hear the tone, please advance the slides.

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Part I of the program focuses on the fluorochrome staining procedure. Part II focuses on examining and reporting fluorochrome-stained smears and includes exercises for the participant. In addition to this audiocassette tape and the slides, the program also includes a booklet that contains supplemental information. The booklet will be referred to periodically throughout the program. You may want to look at it now before proceeding.

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Slide 3:

Just what is "acid-fast" microscopy?

Acid-fast ( A-F) Microscopy is: "the microscopic examination of stained smears for the presence of organisms that retain the primary stain when the smear is decolorized with an acid-alcohol solution." On the left is a smear showing the decolorization step in the basic fuchsin stain procedure. On the right, when the smear is viewed microscopically you can see that the acid fast mycobacteria were not decolorized and retained the fuchsin stain. Most Mycobacterium species are strongly acid-fast.

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Fluorochrome acid-fast microscopy is not only easy to perform and cost effective, but is currently the most rapid procedure for detecting acid-fast bacilli in clinical specimens. It is a rapid method to screen for the most infectious cases of presumed tuberculosis. It provides information to support respiratory isolation of patients and other infection control measures which prevent transmission of disease. Acid-fast microscopy is a useful tool for initiating treatment and monitoring the progress of anti-tuberculous drug therapy. Results of acid-fast microscopy are often used as determinants for performing other laboratory tests such as the nucleic amplification tests for identifying M. tuberculosis in clinical specimens. Because smear results impact heavily on subsequent actions, it is critical that acid-fast microscopy be performed so that maximal sensitivity and specificity are achieved and errors are minimized.

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The biggest drawback to fuchsin acid -fast microscopy is the need to use an oil immersion objective at a high magnification to examine smears. Magnifications of 1000 times are required to detect these small acid-fast bacilli scattered within the stained background of non-acid fast particles and cellular material. Because the microscopic field is so small at these magnifications, 300 fields should be examined before a smear is considered negative. It takes an experienced microscopist about 15 minutes to observe 300 fields at 1000x magnification--a fatiguing and time-consuming undertaking, especially if numerous slides need to be examined. The number of smears that can be examined by a single microscopist within a prescribed time is, therefore, limited. This limitation can cause delays in the turnaround time for reporting smear results. Fluorochrome-stained smears, on the other hand, can be viewed at lower magnifications.

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On the left is a photomicrograph of a fluorochrome-stained smear as viewed at 400 x magnification; on the right, a fuchsin-stained smear as viewed at 1000 x magnification using oil immersion. Note how much easier it is to see the fluorochrome-stained organisms at a lower magnification without the use of oil immersion. When lower magnifications are used, less microscopic viewing time is required. Because less viewing time creates the potential for decreasing the turnaround time needed to report microscopy results, CDC recommends fluorochrome-staining for detecting mycobacteria in smears.

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Let's look at each step of the acid-fast microscopy process as it applies to the successful use of fluorochrome dyes. We'll discuss preparing and fixing smears; staining smears; examining smears and reporting smear results. An example of a method for quantitative recording and reporting smear results will be discussed. Details of the various staining procedures can be found in the accompanying booklet.

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Acid-Fast microscopy can be used to evaluate all types of specimens--sputum and other respiratory specimens, scrapings from lesions, sediments of centrifuged body fluids, and tissues. Both viable and killed organisms will stain acid-fast. To enhance the sensitivity of acid-fast microscopy, sputum and other respiratory specimens are usually decontaminated and concentrated by centrifugation at 3,000 to 3,800 times "g". In some situations the cytocentrifuge may be used to prepare smears for acid fast staining. If you use the cytocentrifuge, specimens such as sputum must be liquefied and decontaminated with sodium hypochlorite to kill any mycobacteria present. Once treated in this manner, the specimen is no longer suitable for culture.

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An important detail of acid-fast microscopy is selecting and labeling the microscope slides on which smears are placed. You should use only new, unscratched, and clean slides; using old, scratched, or dirty slides can lead to erroneous results. Each slide should be clearly labeled with the patient identifier that links the smear to the clinical specimen. Do not put the identifier on the slide with a substance that will bleed or flake during staining. Graphite pencils are good; felt-tipped pens and wax pencils are not. Use a diamond or tungsten carbide stylus on slides without a frosted end. Identifiers must be legible throughout the staining procedure.

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When preparing the smear, keep the area of the smear relatively small. An area of approximately 2 by 1 inch (or 2 cm²) is recommended. Larger areas are self-defeating, decreasing efficiency during microscopic examination. As shown here, you may find using a template helpful.

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Be sure to prepare smears of suitable thickness. Smears that are too thick may flake during staining and may be difficult to decolorize. Acid-fast organisms that might be present may be obscured. Smears that are too thin may not contain enough sample. Either condition--too thick or too thin--can lead to erroneous results, particularly false negatives. Here the smear in the center is of the proper thickness. A simple test for you to use is to hold a smear about 3 to 4 inches over news-print. You should just be able to read the print if the smear is of the proper thickness.

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Here is a smear prepared using the cytocentrifuge. This technique may enhance the preparation of smears from specimens such as CSF, broncho alveolar lavage and other fluids. Follow the manufacturer's recommendations when using the cytocentrifuge. If the cytocentrifuge is used to prepare smears, it should be indicated on the smear report.

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Before heat fixing, smears should be air dried. Use of an electric slide warmer is usually the preferred method for heat-fixing smears. The temperature of the warmer is set at 65 to 75^o Centigrade. Do not exceed 75C. Slides are placed on the warmer and the smear fixed for a minimum of 2 hours. This should be performed in a biological safety cabinet. Wet slides should never be brought outside the biological safety cabinet before they are completely dry.

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Slide 14:

An alternate method of heat-fixing is to pass the dried slide, smear facing upward, 2 to 3 times through the blue cone of a burner flame.

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Fixed smears are stained with a fluorochrome dye such as Auramine O seen here on the left. If stained organisms are present in the smear, the acid-fast bacilli will fluoresce (seen on the right). The appearance of the smear's background and the color of the fluorescing organisms are dependent on the fluorochrome dye and counter stain selected for use.

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Fluorochrome dyes used for acid-fast staining include Auramine O, and Auramine O in combination with another fluorochrome, Rhodamine B. Commonly used counter stains include potassium permanganate and acridine orange.

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An important consideration is the quality of the water used for preparing reagents and rinsing slides. Because smears will be recorded as positive whenever acid-fast organisms are seen, it is important that observed organisms originate with the specimen and are not environmental contaminants. Acid-fast microscopy cannot distinguish between a true pathogen and a contaminant. It is critical, therefore, to prevent the introduction of environmental contaminants into the staining process. Daily use of a negative control smear handled exactly the same as patient specimens will assist in detecting environmental contaminants present in water or staining reagents.

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Water is the most notorious source for environmental acid-fast contaminants. Carboys and other large containers are known breeding grounds for contaminants; avoid using them. You should periodically monitor water filtration and distribution systems for bacteria. Consult references in the accompanying booklet for procedures on quality control of laboratory water. 

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When staining slides, first flood the fixed smear with a phenolic solution of the fluorochrome dye, allow the dye to react, and then rinse the slide with water. Flood the smear with the acid-alcohol solution which will not decolorize acid-fast organisms but will decolorize other organisms. After decolorization, rinse the smears with water and if desired, add a counter stain. Rinse the counter stain from the smear, drain and air-dry the slides.

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You can prepare staining reagents in-house or purchase them commercially. Several different formulations for stains have been used successfully. The formulas are in your booklet.

Fluorochrome dyes and staining solutions should be protected from light when not in use. Because some of the components in the staining solutions may be potentially carcinogenic, wear gloves when preparing the reagents and staining slides. When preparing the stains, weigh the dyes within a fume hood with appropriate ventilation.

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In this next series of slides, we'll look at the staining process in more detail using the fluorochrome, Auramine-0 and the counter stain, potassium permanganate. Note how the slides have been placed on the staining rack. Each slide is positioned so that it does not touch adjoining slides. This separation prevents slide-to-slide transfer of organisms which could lead to cross-contamination and subsequent false-positive results. For the same reason, Coplin jars, staining dishes, or other vessels for bulk staining should not be used. Flood smears with sufficient stain to prevent drying during the designated staining time. Should drying occur, add more stain. Note that both a positive and a negative control slide are also being stained. Include positive and negative controls each day that staining is done, ideally, with each run.

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The positive and negative control slides assess the quality of the staining reagents; they let you know if the staining procedure was done correctly and, if your microscope is working properly. The negative control slide will help you detect environmental contaminants in your staining reagents and rinse water. The positive control slide will help in finding the plane of focus for examining patient smears.

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After the designated staining time, rinse the stain from the slides with water.

In fluorochrome staining, it is important that the water does not contain chlorine. Chlorine can interfere with fluorescence. Use of distilled or deionized water will eliminate any problem that might be caused by chlorine. Drain excess water from the slides so the next reagent is not diluted.

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Destain the smears by flooding the slides with acid-alcohol. After decolorizing for 2-3 minutes, only acid-fast organisms, if present in the smears, will retain the primary dye.

You should be aware that the concentrations of both the acid and the alcohol in the decolorizing solution used for fluorochrome staining are less than the concentrations in the decolorizing solution used for fuchsin-staining; thus, you cannot use the same decolorizing solution for both methods.

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Rinse the slides with water as before to remove the acid-alcohol.

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Flood the smears with the selected counter stain. Here, the counter stain is potassium permanganate. Use of a counter stain however is not absolutely necessary but most microscopists prefer to use a counter stain. When potassium permanganate is used, adherence to the designated staining time is critical.

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Rinse the counter stain from the slides with water. Allow excess water to drain off. Let the smears air-dry without blotting.

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Slide 28:

A fluorescence microscope is required for examining fluorochrome-stained smears. Because fluorescence tends to fade with time, smears should be examined as soon as possible after staining and certainly within 24 hours. If smears cannot be examined immediately after staining, refrigerate them in the dark. Schedule your work so that fluorochrome-stained slides can be examined within 24 hours. If you can not examine slides within 24 hours, you should not be doing fluorochrome acid-fast microscopy.

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This is a microscopic field of a fluorochrome stained smear showing numerous green acid-fast bacilli.

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Usually, an incident-light fluorescence microscope is used for acid-fast microscopy. In this diagram of incidence-light fluorescence, the light supplied by the microscope's light source as indicated by the blue line, first passes through the exciter filter. The dichroic beam-splitting mirror reflects selected wavelengths of light to the surface of the specimen, exciting the dye molecules to fluoresce. The sample absorbs the shorter wavelengths of light and emits the longer wave length, fluorescent light as indicated by the green line. The light then passes through the objective, the dichroic beam-splitting mirror, and the barrier filter to the eyepiece for viewing by the microscopist. Refer to your booklet to see examples of filter sets and the excitation and emission wavelengths used in fluorescence microscopy. This information will assist you in working with the microscope manufacturer in selecting the appropriate filter system for your microscope based on the type of fluorochrome stain you want to use.

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You want to examine each selected microscope field thoroughly when examining smears for acid-fast organisms. When examining fluorochrome-stained smears, you must examine the smears in an orderly manner to ensure that a representative area of the total smear has been observed. Two patterns for field selection are shown here. Either is satisfactory, but each microscopist in your laboratory should be consistent in the way he or she examines a slide so that reliable results are obtained from day to day.

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As you can see in this chart, the greater the magnification, the more microscopic fields you need to examine. At 200x and 250 x magnification, at least 30 fields should be examined before a smear is considered negative. At a magnification of 400x, 50 to 55 fields should be examined.

Total magnification is determined by multiplying the magnification of the objective by the magnification of the eyepiece. Thus, to achieve a total magnification of 200x, you would use a 20x objective and a 10x eyepiece. Magnifications below 200x are not recommended because, at the lower magnifications, the intensity of fluorescence is so minimal that the small acid-fast bacilli may be difficult to see.

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This chart, also found in the booklet, shows a numerical scheme for reporting results. For example, if no organisms are seen after examining 30 fields at 200x or 250 x, the report is "No Acid-fast Organisms Seen." A report of negative for acid-fast bacilli should never be used.

If only a few organisms are seen and results are questionable, the test should be repeated.

A reading of 2+ indicates that 1-9 organisms are seen in every field at 200-250x or 4-36 organisms are seen in a total of 10 fields at 400-450x. When you report your acid-fast microscopy results, always state the staining method you used, and the number of acid-fast bacilli you observed per number of fields.

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Acid-fast microscopy results should be reported to the clinician within 24 hours after the laboratory receives the specimen. Use the telephone or some other form of electronic method of communication to rapidly transmit the results. Results transmitted orally must be followed by a written report.

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Achieving reliable results depend on obtaining quality specimens; and ensuring that the specimen or testing sample does not become contaminated. Laboratory practices must be such that no mechanism for transfer of organisms from other testing samples or the environment exists. It is essential to follow established procedures and recommendations and keep accurate records so that the results you report correlate accurately with the identity of the patient and the original specimen.

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Achieving reliable results depend on obtaining quality specimens; and ensuring that the specimen or testing sample does not become contaminated. Laboratory practices must be such that no mechanism for transfer of organisms from other testing samples or the environment exists. It is essential to follow established procedures and recommendations and keep accurate records so that the results you report correlate accurately with the identity of the patient and the original specimen.

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By following the procedures and guidelines presented in this program, you can achieve reliable results when performing fluorochrome-staining for acid-fast bacilli. This completes Part I of the program. You may now proceed to Part II, which will give you practice in examining and interpreting fluorochrome-stained smears. The audio for Part II is on the other side of this cassette.
  

This is Part II of the program, Use of Fluorochrome Acid-fast Staining for Detecting Mycobacteria. This part focuses on examining, interpreting and reporting fluorochrome stained smears. A relatively dark room should be used to ensure optimal visibility of acid-fast organisms in the photomicrographs in this part of the program. You will need to refer to the chart "Examining And Reporting Acid-fast Smears", in your booklet. Please turn to it now. (PAUSE)

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First we will look at the combinations of colors of the acid-fast organisms and background of smears stained with different fluorochrome procedures and counter stains. The color of acid-fast organisms stained with these fluorochromes may vary slightly depending on the microscope filter system used. Refer to the table and references in your program booklet for further information on how filters are selected for these stains. When Auramine O is the primary stain, acid-fast organisms will appear yellow green. With the combination of Auramine O and Rhodamine B as the primary stain, the organisms will appear yellow orange. When potassium permanganate is used as the counter stain, fluorescence is quenched and the background appears dark. If acridine orange is used as the counter stain, background material such as bacteria, yeasts and other cellular debris will stain yellow orange. Now let us look at smears stained with the combinations of primary stains and counter stains.

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Slide 3:

This smear has been stained with Auramine O and the counter stain potassium permanganate. Here, the acid-fast organisms appear green on a dark background. Numerous acid-fast organisms are present. Because we are looking at only one plane of focus, some organisms will not appear as green as others. In this 400x magnification, note that the size and staining characteristics of the acid-fast organisms vary. (PAUSE)

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This smear was stained with Auramine O and counter stained with Acridine Orange. Notice the Acridine Orange stains the epithelial cells in the specimen yellow to orange. The acid-fast organisms are green. How many acid fast organisms do you see here ? (PAUSE) You should be able to see at least 12. Starting at 1 o'clock, there are 2 organisms, then at 3 o'clock there are 3. Continuing clockwise to the 7-9 o'clock area you can see an additional 4 acid-fast organisms and at 12 o'clock, 2 organisms. Don't forget to observe the center of the field. Notice that some of the organisms are very thin and are not as easily seen as others.

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Here, the primary stain is a combination of Auramine O-Rhodamine B and the counter stain is potassium permanganate; magnification is 400x. Notice that in this smear the organisms may appear varying shades of green and yellow. Some even appear gold. Acid-fast organisms may display a diverse morphology. Notice that some of the acid fast bacilli show a beading effect. This can be seen in the bacilli at 6 and 9 o'clock. Also notice the thin slender rods, some of which are curved. Curved acid-fast organisms can be seen at 6, 7 and 12 o'clock.

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Let's compare the appearance of a smear examined at 400x magnification vs. one examined at

200x magnification. Note the difference in size of the acid-fast organisms at 400x magnification on the left and the 200x on the right. Notice also the difference in the size of the epithelial cells. Some experienced microscopists may screen at 200x and switch to the higher 400x magnification to confirm the presence of acid-fast organisms. The magnification selected should be optimal for the microscopist to detect the presence of acid fast organisms. Smears should not be read at less than 200x magnification. You will see later in this presentation that magnification is not only important in detecting acid-fast organisms but also in determining the number of fields to be examined.

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In fluorochrome microscopy, positive and negative control slides must be stained each time patient smears are stained. Control slides should be examined before observing patient smears. The controls determine the validity of the staining procedure and may assist in confirming that the microscope is properly adjusted. Note the green color of the organisms in the positive control on the left. This is, of course, an Auramine O stain with Potassium Permanganate used as the counter stain. Look at the negative control on the right. Note the nonspecific fluorescence in the background of the smear. It is very important to always carefully observe the negative control slide. The appearance of an acid-fast organism in the negative control indicates a potential environmental contaminant. The program booklet contains references on preparing positive and negative control slides. To help you to gain experience in interpreting acid-fast smears, we will be examining a set of slides, and you will evaluate these smears and report the results. For this part of the program, you will need to refer the chart in your program booklet " Examining And Reporting Acid-fast Smears". (PAUSE). Notice in the chart that magnification, number of acid-fast organisms seen per field, and the number of fields examined are all important criteria in interpreting and reporting acid-fast smear results.

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This smear was stained with Auramine O using an Acridine Orange counter stain and viewed at 400x magnification. Observe that background debris and autofluorescence are prominent in this field. Note the presence of the green acid fast organisms. How many organisms can you see? (PAUSE). Starting at 1 o' clock and progressing clockwise, you should see at least 12 acid-fast organisms. Do you see at least 12? Let us assume that this field is representative of the first 10 fields examined on an acid-fast smear. How would you report the results of the smear? Refer to the chart. According to the chart, the smear would be reported as: Positive for acid-fast organisms, (3+) Remember you counted at least 12 organisms at 400x magnification in 10 fields. Studying the chart, you see this is within the range of 4-36 organisms /10 fields and is equivalent to 3+ . Are you ready for the next slide?

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Observe this field for acid-fast bacilli. What is the primary stain? What is the counter stain? (PAUSE). If you said Auramine O with Potassium Permanganate, you are correct. 

Now, lets help you a little more. One acid fast bacillus in the center of the field is obvious. In the lower left of the field, 2-3 possible organisms are seen. Remember, fluorochrome stained smears can be re-stained with a fuchsin method; the reverse, however, is not true--fuchsin-stained smears cannot be re-stained with a fluorochrome stain. If only 1-2 acid fast organisms are seen after observing more than 70 fields at 400x or 450x magnification, the smear should be reported as "equivocal or doubtful" and should be repeated. Another smear should be prepared, stained and evaluated from the same specimen or from another specimen if possible.

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Observe the organisms in this field at 400x magnification. What is the primary stain used here? You are right, if you said Auramine O stain with Acridine Orange as the counter stain. Note the yellow stained epithelial cells. Some cells retained more stain than others. If a smear retains a lot of stain over several areas of the slide, acid-fast organisms may not be seen and the smear may be improperly interpreted. That is not the case in this field. You can see several acid-fast organisms; some are touching each other as seen at 5 o'clock and in other areas of this field. When organisms are touching or clumping they are counted as one organism because they form one colony forming unit when they grow. You can see several acid-fast bacilli lying on top of the epithelial cells. In this field, more than 36 organisms are seen, therefore if every field you examine contains the same number of organisms seen here, that is at least 36, how would you interpret this smear? (PAUSE). Remember refer to the chart at the magnification of 400x. The number of acid-fast organisms in this field is greater than 36 and all fields contain approximately the same number, therefore the report would be: Positive for Acid fast organisms, 4 +.

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This is a 400x magnification of an Auramine O stain with Acridine Orange counter stain. Count the number of acid-fast organisms here. (PAUSE) You should see at least 18 organisms. How would you report this smear if the number of organisms seen here were found in each field examined? This smear would be reported as: Positive for acid-fast organisms; 3+ since there are at least 18 acid-fast bacilli /field.

This an Auramine O stain with Acridine Orange counter stain. The left side of the smear is a 200x magnification. The right side is 400x. Note the large number of yellow or orange bacilli. Remember that acid-fast organisms stained with the Auramine O stain should appear green and background debris will stain yellow to orange. Do you see any green acid-fast organisms here? You should see 2 or 3 acid-fast organisms on the left at 200x magnification and 2 on the right at 400x. How would you report this smear if you observed 10 fields and 3 acid-fast organisms were seen in each field at 200x magnification? The quantity would be equivalent to 1+. Note in the chart that a 1+ represents 1-9 acid-fast organisms seen in 10 fields at a magnification of 200x. Observe also in your chart that at a magnification of 400x, 2-18 acid-fast organisms per 50 fields are equivalent to 1+. Therefore, the number of organisms counted per number of fields observed at a specific magnification is very important in reporting acid-fast smear results. As you can see, the majority of the organisms in this field appear non- acid fast. Therefore, smears must be examined very carefully when there is a dense background of bacteria or other organisms present and the morphology of acid-fast organisms should be confirmed at 400x magnification.

This completes Part II of the Program on examining and reporting fluorochrome-stained smears. Consult your program booklet for additional references and resources and take the quiz to assess the knowledge you have acquired from material covered in both Part I and II of this program.