Staining of Supplied Smears: 2 Methods

The following points highlight the two methods of staining of supplied smears. The methods are: 1. Albert’s Stain 2. Ziehl-Neelsen (ZN) or Acid-Fast Staining.

Method # 1. Albert’s Stain:

Usually examinee is provided with a fixed smear prepared from C. diphtheriae grown usually on Loeffler’s serum slope or occasionally from blood tellurite agar medium (Fig. 4.1C). If it is an unfixed smear, fix it by heating gently by passing it over a flame several times. Then proceed it for staining.

Method:

1. Microscopic examination of stained smear is done using x 100 lens (oil immersion object­ive) with condenser up.

2. Show the examiner, a well stained area focu­sed under the microscope. Importance is given on quality of stain.

Observations:

Bacillary body and metachromatic granules of Corynebacterium diphtheriae, take green and bluish black colours respectively. Arrangement shows angular patterns of V, X, Y, H etc., often described as cuneiform or Chinese letter. Draw a faithful diagram of focused field and label it (see Fig. 3.1).

Corynebacterium:

Corynebacteria are pleomorphic, Gram-positive, non- acid-fast, non-sporing, non-motile bacilli measuring about 3 μm x 0.3 μm. They often show club-shaped swellings (Koryne — club).

Classification:

A. Pathogenic:

(a) C. diphtheriae, classic model of bacteria, is the causative agent of diphtheria.

(b) Other pathogenic corynebacteria: These are primarily animal pathogens. C. ulcerans cau­sing acute pharyngitis, C. pseudo tuberculosis causes chronic lymphadenitis in axilla and C. jeikeium is an opportunistic pathogen in man.

B. Non-pathogenic:

C. Xerosis—a commensal of conjunctival sac and C. hofmannii (C. pseudodiphtheriticum), a commensal of oral mucosa.

Corynebacterium Diphtheriae:

There are three subspecies of C. diphtheriae:

1. C. diphtheriae gravis with 13 serotypes.

2. C. diphtheriae intermedius with 4 serotypes.

3. C. diphtheriae mitis with 40 serotypes.

Gravis type is associated with severe form, intermedius type with intermediate form, and mitis type with mild form of diphtheria. Infection due to C. diphtheriae mitis is common in India.

Diphtheria toxin:

The production of exotoxin depends on the presence of a tox gene in chromosome of lysogenic C. diphtheriae. The toxin is a heat-labile polypeptide (MW 62,000) and consists of two fragments — A and B — linked by a disulphide bridge. Both fragments are essential for cytotoxicity.

The amino terminal portion of the toxin, fragment A (21 KDa), contains the enzymatically active site of the toxin. Fragment B (37 KDa) binds the toxin to eukaryotic cell.

The toxin is thought to inhibit protein synthesis of host cell acting at the ribosomal level by splitting the molecule of NAD (nicotinic adenine dinucleotide) — an essential co-factor for the transferase involved in peptide bond formation by ribosomes.

The toxin can be rendered non-toxic but still antigenic by treatment with formaldehyde and application of heat. Toxoid thus formed is used in prophylactic immunization.

Pathogenesis:

1. Local effect:

The bacteria remain localised at the site of entry (usually upper respiratory tract) and multiply on the mucous membranes and produce exotoxin. The toxin causes necrosis of the mucosal cells along with superficial inflammatory reaction. The necrosed epithelium together with fibrinous exudate, leucocytes, red cells and bacteria form a grey, adherent pseudo membrane.

2. General effect:

After absorption into blood stream, the toxin acts systematically on the cells of the myocardium, nervous system (only motor nerves) kidney and adrenals.

Laboratory diagnosis:

Patient suspected of having diphtheria is treated immediately with antitoxin without waiting for laboratory report as the toxin is powerful and rapidly fatal.

Specimens:

Swabs from throat, nose or larynx depending on the site of lesion. Membrane may be found in skin ulcer or inside vagina.

Microscopy:

Albert-stained smears show long, thin and curved greenish rods arranged in Chinese letter patterns, often beaded due to presence of dark staining volutin granules in the rods. Nonpathogenic C. xerosis also contains sparse number of volutin granules.

The volutin granules are best demonstra­ted by Albert’s staining method in film preparations from luxuriant media (e.g. Loeffler’s serum me­dium). The morphological similarity of C. diphtheriae and C. xerosis demands that they should be differ­entiated by further tests.

Culture:

The material is inoculated in:

(1) Loeffler’s inspissated serum medium and

(2) Tellurite blood agar medium and incubated at 37°C for 12 hours and 24 hours, respectively.

(3) When the specimen is inoculated in blood agar colonies appear after 24-48 hours incubation. Albert’s staining of smear made from colony is examined to see the bacterial morphology.

Biochemical reaction:

Isolated organism is inoculated in Hiss’s serum medium containing sugar (glucose, maltose, sucrose, mannitol). The toxigenic strains usually produce acid in glucose and maltose, whereas the diphtheroids ferment sucrose and mannitol.

Toxigenicity test:

1. Guinea pig inoculation:

Suspension of isolated strain of C. diphtheriae (0.3 ml) is injected subcutaneously into thigh of two guinea pigs, one protected intramuscularly with 500 units of diphtheria anti­toxin 18-24 hours before the test.

Observation:

The unprotected animal dies within 2-3 days with evidence of haemorrhage in the adrenal glands.

2. Gel-precipitation (Elek) test:

A rectangular strip of filter paper previously incorporated in diph­theria antitoxin (1,000 units/ml) is incorporated into serum agar plate before it has set. The testing strain is then streaked across the plate at right angles to the filter paper strip and incubated at 37°C for 24-48 hours.

Observation:

The lines of precipitation radiate a little distance away from the intersection of the strip and bacterial growth.

Typing:

The isolated strain of C. diphtheria is further subdivided by serotyping by agglutination tests, phage typing and bacteriocin typing for epidemio­logical studies.

Method # 2. Ziehl-Neelsen (ZN) or Acid-Fast Staining:

Generally, a fixed smear is supplied in examination as stress is made mostly on the staining procedure. If, the smear is unfixed, fix it by gently heating the smear on flame.

Method:

Examine the stained smear under oil- immersion objective (x 100) of microscope.

Observations:

1. Mycobacterium tuberculosis is seen as bright red bacilli (AFB). See Fig. 3.3.

2. Draw a labelled diagram of your findings and focus under microscope.

Mycobacterium:

Mycobacteria are non-motile, non-sporing, non- capsulate, slender acid-fast bacilli and sometimes exhibit filamentous forms resembling fungal mycelium (Greek Myces meaning fungus) and, hence, they are so named. Hansen (1868) discovered the first member (Lepra bacillus), Robert Koch (1888) isolated mammalian tubercle bacillus, and Johne (1895) described M. Para tuberculosis (Johne’s bacillus).

Mycobacterium species:

The genus Mycobacterium contains over 50 well-defined species. In developing countries, the major pathogenic species are M. tuberculosis, M. bovis and M. leprae. A tentative classification of those species capable of producing human disease are listed in Box 4.

Mycobacterium Tuberculosis:

Habitat:

The main reservoirs of M. tuberculosis are infected persons, also occasionally present as patho­gens in animals.

Pathogenesis:

Tuberculosis is a slowly progressive, chronic gra­nulomatous infection. Mycobacterium tuberculosis is the main causative agent and less often Mycobacte­rium bovis. Clinically tuberculosis is seen in two forms: primary and post-primary infections.

I. Primary Infection:

Primary infection may occur in any organ such as lung, tonsils/intestine or skin, but in children the usual site is lung and is called primary complex.

Primary complex:

This consists of:

(i) A small local lesion as peripheral or sub pleural focus of tuber­culosis pneumonia in the lung parenchyma (Ghon focus),

(ii) Enlargement of the regional hilar lymph nodes, and

(iii) Lymphangitis of the draining lymph vessel. In most cases the primary complex heals spontaneously.

It may, however, progress to tuber­culous bronchopneumonia, miliary tuberculosis, tuberculous meningitis, bone and joint tuber­culosis and genitourinary tuberculosis (renal, endometrial, epididymis).

II. Post-primary infection:

The lesions, chara­cteristically more localised and fibrotic, are modified by the development of hypersensitivity by the host.

Source:

(i) Endogenous:

Mainly due to reactivation of primary lesion.

(ii) Exogenous:

Reinfection by bacilli newly inha­led or ingested from the environment.

Lesions:

It most often involves the lung with lesions in the apical region. In untreated case, chronic progressive disease develops with tubercle formation, caseation, cavitation and shedding of bacilli.

Immuno compromised patients (ICP):

ICPs, especially those with HIV infection, are extremely susceptible to tuberculosis. The infection may be rapid and overwhelming with depression of delayed hypersensitivity.

Laboratory diagnosis:

Specimen:

1. Sputum – concentrate prepared.

2. Urine – 3 consecutive morning samples used, concentrate prepared.

3. CSF – centrifuged aseptically before culture.

4. Bone, joint, endometrium samples — disrupted in a sterile grinder before culture and concentrate prepared.

Microscopy:

Slender, 2-3 x 0.2-0.4 μm, non- sporing, beaded bacilli. Smear is stained by Ziehl- Neelsen stain. Heat-fixed smears are treated with concentrated carbol fuchsin, mordanted by heating and then decolorized with 20% sulphuric acid and alcohol and counter stained by methylene blue.

The bacilli retain bright red colour of carbol fuchsin against the background of counter stain. For positive microscopic finding, there should be at least 20,000 bacilli per ml of sputum. Nowadays, AFB is usually detected in Western countries by fluorescent microscopy with aura-mine stain. It facilitates rapid diagnosis and is useful in mass screening.

Culture:

M. tuberculosis is an obligate aerobe and does not grow on ordinary media. It grows luxu­riantly (eugonic growth) on Lowenstein-Jensen medium which contains egg, asparagine, glycerol and malachite green. Glycerol augments growth of M. tuberculosis and inhibits that of M. bovis.

The malachite green inhibits contaminants, but contami­nated specimens such as sputum, laryngeal swabs, gastric juice, pooled specimens of urine and pus from secondarily infected wounds require deconta­mination and concentration of the specimen before culture. For routine purposes, sodium hydroxide (4% W/V) is an effective decontaminant.

Concentration of specimen:

Concentration method not only increases the organismal popu­lation per unit volume of sample but also kills the contaminated organisms other than mycobacteria. Concentrate of specimen is used for smear, culture and animal inoculation. Culture is a more sensitive method for detection of bacilli, moreover, it is necessary for drug sensitivity.

1. Petroff’s method:

About an hour before culturing, mix equal volumes of sputum and 4% sodium hydroxide and incubate at 37°C for 20-30 minutes. Shake at intervals to homogenize the sputum.

After centrifugation at 3,000 rpm for 15-20 minutes, it is neutralized with 8% hydrochloric acid in presence of phenol red indicator. A “wide range indicator paper” may be used. After neutralisation, the deposit is again washed with sterile normal saline. The supernatant is thrown-off.

2. Acid homogenization method:

Equal volumes of specimen and 6% sulphuric acid are mixed. The mixture is then centrifuged. The deposit is washed three times with sterile normal saline.

3. NALC method:

0.5G N-acetyl-L cysteine (NALC) in 50 ml 1 N (4%) NaOH and 50 ml 0.1 M (2.94%) trisodium citrate, 3 H₂O digestant is mixed (by vortexing) to sputum in equal volume (sputum: digestant = 1:1) and allowed to act for 20 minutes followed by addition of 0.67 M phosphate buffer (pH 6.8) in excess amount.

The whole thing is centri­fuged. The deposit is re-suspended in 1 ml of phos­phate buffer (pH 6.8) with bovine serum albumin (BSA). This re-suspended material is used for culture and smear preparation.

The concentrate of specimen thus prepared is utilised for preparation of ZN stained smear and also for culture. By this technique, larger number of cases of tuberculosis can be detected. Smear examination sensitivity is 6000-10,000 bacilli/ml.

Inoculation and incubation:

Concentrate of sputum, urine, tissue and deposit of CSF are cultured in two bottles of Lowenstein-Jensen medium and incubated at 37°C in dark and light up to 8 weeks. Cultures are examined first after 4 days for rapid growing mycobacteria and then weekly till 8 weeks. Tubercle bacilli usually grow in 2-8 weeks. Longer incubation up to 12 weeks may be necessary for strains originating from treated patients.

M. tuberculosis often grows as twisted rope-like colonies in liquid culture medium which are called serpentine cords. This cording of the organism is not specific for M. tuberculosis and may occur to a lesser extent with other Mycobacterium species.

Identification of M.tuberculosis cultures:

1. Colonies:

Dry raised, yellowish colonies in LJ medium, often described as “rough, tough and buff”.

2. Morphology:

ZN stained smear of culture shows slender, beaded, acid and alcohol-fast bacilli. Many M. tuberculosis strains show cording (serpentine cords). See Table 3.1.

3. Niacin test:

M. tuberculosis is niacin positive.

Animal inoculation:

Part of the concentrated material (0.3 ml) or 0.5 ml of culture suspension is inoculated intramuscularly into thigh of a tuberculin negative 12-week-old healthy guinea pig.

The animal develops dissemi­nated disease in 4-8 weeks and becomes tuberculin positive. Post-mortem findings include caseous lesion at inoculation site, caseous draining lymph node, tubercles in lungs, spleen and kidney. M. tuberculosis is virtually non-pathogenic for rabbits.

Nucleic acid amplification:

Molecular techni­ques like genetic probes and PCR are now increas­ingly used in developed countries for rapid diag­nosis of tuberculosis. The sensitivity of the test ranges from 75-100%.

Atypical mycobacteria:

Several species of myco­bacteria other than Mycobacterium tuberculosis (MOTT) can infect humans (Table 3.2).

Vaccine:

Bacille Calmette-Guerin (BCG) vaccine, an alternated strain of M. bovis, when injected intra- dermally, it confers tuberculin hypersensitivity and enhanced activity to macrophages that kill the pathogen. It is about 80% protective in serious forms of tuberculosis such as meningitis in children as has been used in mass immunization by WHO.

Staining Blood Film:

Blood parasites such as plasmodia etc. are studied by Leishman’s stain and Giemsa’s stain.

Leishman’s stain:

Stain may be purchased ready for use (0.15 G of Leishman’s powder dissolved in 100 ml of pure alde­hyde-free methyl alcohol).

Staining technique:

Pour undiluted stain on air dried unfixed blood film and allow to act for one minute. Then add double the volume of distilled water on the slide by means of a pipette, mix and leave for 10 minutes. Wash slide gently with water and dry. Examine under oil immersion lens. Malaria parasites are demonstrated by this method.

Giemsa’s stain:

Stain is prepared by mixing different proportions of methylene blue and eosin. Ready-made stain is commercially available. It is a useful stain to demon­strate malaria parasites, Schuffner’s dots and Trypanosomes.

Staining technique:

(1) Fix films in methyl alcohol for 3 minutes.

(2) Stain in a mixture of 1 part stain and 10 parts buffer solution pH 7.2 for one hour.

(3) Wash with buffer solution, allowing the preparation to differentiate for 30 seconds.

(4) Blot and dry in the air.