The cell walls of mycobacteria contain long-chain, multiply cross-linked fatty acids, called mycolic acids. Mycolic acids probably complex basic dyes, contributing to the characteristic of acid-fastness that distinguishes mycobacteria from other bacteria. Mycobacteria are not the only group with this unique feature. Species of Nocardia and Rhodococcus are also partially acid-fast; Legionella micdadei, a causative agent in pneumonia, is partially acid-fast in tissue. Cysts of the genera Cryptosporidium and Isospora are distinctly acid-fast. The mycolic acids and lipids in the mycobacterial cell wall probably account for the unusual resistance of these organisms to the effects of drying and harsh decontaminating agents in addition to the property of acid-fastness.

When Gram stained, mycobacteria usually appear as slender, poorly stained, beaded, gram-positive bacilli (Figure 1); sometimes they appear as “gram neutral,” or “gram-ghosts,” by failing to take up either crystal violet or safranin. Acid-fastness is affected by the age of colonies, the medium on which growth occurs, and exposure to ultraviolet light. Rapidly growing species appear to be acid-fast variable.

Fig1. Gram staining of M. marinum demonstrates beaded appearance. (Courtesy Stacie Lansink, Sioux Falls, S.D.)
Three types of staining procedures are used in the laboratory for rapid detection and confirmation of acid-fast bacilli: fluorochrome, Ziehl-Neelsen, and Kinyoun. Smears for all methods are prepared in the same way.

Visualization of acid-fast bacilli in sputum or other clinical material should be considered only presumptive evidence of tuberculosis, because staining does not specifically identify M. tuberculosis. The report form should indicate this. For example, M. gordonae, a nonpathogenic scotochromogen commonly found in tap water, has been a problem when tap water or deionized water has been used in the preparation of smears or even when patients have rinsed their mouths with tap water before using an aerosolized saline solution to induce sputum. However, the incidence of false-positive smears is very low when good quality control is maintained. Conversely, acid-fast stained smears of clinical specimens require at least 10^4 acid-fast bacilli per milliliter for detection from concentrated specimens.
Methods Fluorochrome Stain. Fluorochrome staining is the screening procedure recommended for laboratories that have a fluorescent (ultraviolet) microscope. Fluorochrome stain is more sensitive than the conventional carbolfuchsin stains, because the fluorescent bacilli stand out brightly against the background (Figure 2). Because the smear can be examined initially at lower magnifications (×250 to ×400), more fields can be visualized in a short period. In addition, a positive fluorescent smear may be restained using the conventional Ziehl-Neelsen or Kinyoun procedure, thereby saving the time needed to make a fresh smear. Screening of specimens with rhodamine or rhodamine-auramine results in a higher yield of positive smears and substantially reduces the time needed to examine smears.

Fig2. M. tuberculosis stained with (A) fluorochrome stain (×400) and (B) Kinyoun acid-fast stain (×1000).

One drawback of the fluorochrome stains is that many rapid-growers may not appear fluorescent with these reagents. All positive fluorescent smears should be confirmed with a Ziehl-Neelsen stain or by examination by another technologist. It is important to wipe the immersion oil from the objective lens after examining a positive smear, because stained bacilli can float off the slide into the oil, possibly contributing to a false-positive reading for the next smear examined.

Fuchsin Acid-Fast Stains. The classic carbolfuchsin stain (Ziehl-Neelsen) requires heating of the slide for better penetration of the stain into the mycobacterial cell wall; hence, it is also known as the hot stain procedure. With Ziehl-Neelsen staining, Mycobacterium spp. appear red or have a red-blue, beaded appearance, whereas nonmycobacteria appear blue.

Procedure 1, describes the Kinyoun acid-fast stain. The method is similar to Ziehl-Neelsen staining, but no heat is used (see Figure 2); this technique is known as the cold stain procedure. If present, typical acid-fast bacilli appear as purple to red, slightly curved, short or long rods (2 to 8 µm); they also may appear beaded or banded (M. kansasii). For some nontuberculous species, such as M. avium complex, they appear pleomorphic, usually coccoid.

Procedure 1.

 Examination, Interpretation, and Reporting of Smears. Before a smear is reported as negative, it should be examined carefully by scanning at least 300 oil immersion fields (magnification ×1000), equivalent to three full horizontal sweeps of a smear that is 2 cm long and 1 cm wide. Because the fluorescent stain can be examined using a lower magnification (×250 or ×450) than that required for a fuchsin-stained smear, the equivalent number of fields (30) can be examined in less time, which makes the fluorochrome stain the preferred method.
When acid-fast organisms are observed on a smear, the report should include information about the type of staining method used and the quantity of organisms. The recommended interpretations and ways to report smear results are shown in Table 1.

Table1. Acid-Fast Smear Reporting

The overall sensitivity of an acid-fast smear ranges from 20% to 80%. Factors such as specimen type, staining method, and culture method can influence the acid fast smear sensitivity. In general, specificity of acid-fast smear examination is very high. However, cross contamination of slides during the staining process and use of water contaminated with saprophytic mycobacteria can lead to false-positive results. Staining receptacles should not be used; acid-fast bacilli can also be transferred from one slide to another in immersion oil. For these reasons, the best course is to confirm a positive result.

Although not without some limitations, because of its simplicity and speed, the stained smear is an important and useful test, particularly for detection of smear-positive patients (“infectious reservoirs”), who pose the greatest risk to others in their environment.

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم شؤون المعارف ينظم دورة متقدمة في تحقيق المخطوطات لملاكاته

قسم الشؤون الفكرية يصدر العدد العشرين من مجلة دراسات إفريقية

المجمَع العلمي يكرم الأساتذة المشاركين في مشروع الدورات القرآنية الصيفية في كربلاء

العتبة العباسية المقدسة تختتم فعاليات البرنامج المركزي الأسبوعي لمنتسبيها

المزيد

الآخبار الصحية

هل من الآمن شرب القهوة على معدة فارغة؟

تحذير: مرض السكري يسرق طاقة قلبك

حقيقة صادمة.. لماذا يجب أن تتوقف عن شراء الجزر الصغير؟

بعد عمر الستين.. توقيت وجبة الإفطار مؤشر على صحتك

المزيد

الآخبار التكنلوجية

زلزال أفغانستان يحصد أكثر من ألف قتيل.. ويدمر آلاف المنازل !

ناجٍٍ وحيد من انزلاق أرضي.. دمّر قرية سودانية بأكملها !

نهر الفرات.. مورد مائي بين الندرة والتقاسم

ما السر وراء مشية إيلون ماسك الغريبة والسريعة ؟

المزيد

مواضيع ذات صلة

Conventional Phenotypic Tests for Mycobacterial Infections

Genetic Sequencing and Nucleic Acid Amplification for Mycobacterial Infections

Vibrio Parahaemolyticus and Vibrio vulnificus

Vibrio Cholerae

Burkholderia cepacia Complex

Burkholderia pseudomallei and Burkholderia mallei

Rapidly Growing Nontuberculous mycobacteria (RGM)

Slow-growing Nontuberculous Mycobacteria

Mycobacterium Tuberculosis Complex

Anaerobic Gram-Positive and Gram-Negative Cocci

The Salmonellae

The shigellae