Morphological Study of Bacteria: 2 Methods

The following points highlight the two main methods of morphological study of bacteria. The methods are: 1. Unstained Wet Mount 2. Stained Films.

Method # 1. Unstained Wet Mount:

Drops of liquid specimens of 3-6 hours growth in fluid media at room temperature (22°C) are exami­ned mainly for study of bacterial motility. The method is also used for demonstration of spirochaetes from clinical materials by D.G.I.

Hanging Drop Preparation (Figs. 2.1-2.4):

Procedure:

(a) Four beads of plasticin or vaseline is placed over the middle part of a clean glass slide (Fig. 2.1).

(b) With the help of sterile bacteriological loop place a large drop of young broth culture at the centre of a clean cover glass (Fig. 2.2).

(c) Glass slide with adherent plasticin/vaseline beads is inverted gently on the cover glass with culture drop. This will make the cover glass adherent to the slide.

(d) Then the glass slide with adherent cover glass is reverted making the culture drop hanging from cover glass (Fig. 2.3).

(e) The hanging drop is then examined, first under low power (x 10) to focus the periphery of the drop, and then under high power (x 10) to note the motility (Figs. 2.4 and 2.5).

[Caution: Elevation of beads should not be too high to interfere focusing and too little allowing drop to touch the slides. A dry drop is unsuitable for examination].

Observation:

(a) Non-motile bacteria are either static or show Brownian movement, i.e., in different directions without any definite propagative movement, due to repulsion by the surface charge of adjacent bacteria.

(b) True motility is indicated by long distance propagation of bacteria with a definite direction, often away from the field.

(c) Patterns of motility: Swinging, tumbling, sudden bending, spiral, darting, slow gliding etc.

Method # 2. Stained Films:

Routine staining technique of bacteria employs smear preparation, drying, fixation and staining of smears:

1. Smear:

The technique consists of spreading a thin film made from a liquid suspension of cells on a slide and air drying the film.

2. Fixation:

The air dried smear is usually heated gently by passing through the flame.

3. Staining:

By staining procedures, coloured chemicals (dyes) impart colour to the cells or cell parts through a chemical reaction by affixing to them. Smear made from bacterial cultures or specimens are first heat-fixed. Heat kills and fixes the bacteria on slide after coagulating bacterial protein. The fixed film is stained by appropriate dyes.

By ordinary staining method (e.g. simple stain or Gram’s stain) bacterial cell wall cannot be stained. The coloured body seen under light microscope represents cell protoplasm only. In the chain of bacteria, the coloured bacterial bodies are separated by gaps which represent unstained connecting cell walls.

Common staining techniques:

1. Simple stains:

Watery solution of a single basic dye such as methylene blue or basic fuchsins are used as simple stain.

2. Negative staining:

Bacteria are mixed with dyes (India ink or nigrosin). The background gets stained leaving the bacteria contrastingly colourless. The technique is useful in demon­stration of bacterial capsule.

3. Impregnation methods:

Bacterial cells and appendages that are too thin and delicate cannot be seen under ordinary microscope. These delicate structures are thickened by impregnation of silver (special stains) on the surface to make them visible under light microscope, e.g. demonstration of spirochaetes and bacterial flagella.

4. Differential stains:

They impart different colours to different bacteria or their structures. In a stained film, bacterial shape, arrangement and presence of other cells (pus cells) are noted. The three commonly used differential stains are Gram’s stain, Acid-Fast stain and Albert’s staining.

i. Gram’s Stain:

It is the most widely used stain in medical bacte­riology. The stain was originally devised by Chri­stian Gram (1884) as a technique of staining bacteria in tissues. There are four steps in the technique. Bacterial culture in plate or tube is provided to students.

Preparation of smear:

(a) Using a sterile bacteriological loop, a drop of sterile normal saline is placed on a clean grease free slide.

(b) Pick the tip of a bacterial colony on solid me­dium with loop or straight wire, and make a small emulsion with saline. Next spread it into a thin smear.

(c) Air dry the smear.

Fixation of smear:

Dried smear is fixed by passing it several times over a flame. Gentle heating kills the bacteria and coagulation of protein fixes it on slide. Care is to be taken not to overheat to char the bacteria.

Staining Method:

1. Primary staining of heat fixed smear of speci­men or bacterial culture is made with a pararosaniline dye, e.g. crystal violet, gentian violet or methyl violet solution for one minute. Usually the smear is fully covered with crystal violet solution.

2. Pour off crystal violet and add dilute solution of iodine, such as Gram’s iodine—keep for one minute.

3. Wash with water.

4. Decolourisation with an organic solvent (alcohol or acetone or iodine-acetone 1:1 mix­ture) — 10 to 30 seconds till faint blue colour persists.

5. Wash with water quickly to avoid over-decolourisation.

6. Counterstain with a dye of contrasting colour 1: 20 dilute carbol fuchsin, safranin or neutral red for 1/2 to 1 minute. Safranin is commonly used as counterstain.

7. The smear is washed with water and then air or blot dried.

8. The smear is examined under oil-immersion objective (x100) with condenser up.

Observation (Fig. 2.7):

Gram-positive bacteria resist decolourisation and stain violet. Gram-negative and other cells (pus cells) are decolorized and stain pink with counterstain. The Gram-positive bacteria may sometimes appear Gram-negative under certain situations, such as in ageing cultures and when the cell wall is damaged.

On the basis of Gram’s staining, bacteria are divided into two categories:

1. Gram-positive bacteria:

All cocci except Neis­seria, Moraxella and Branhamella are gram- positive.

2. Gram-negative bacteria:

All bacilli are Gram- negative except Corynebacterium, Mycobact­erium, Bacillus, Clostridium, Actinomyces, Listeria, Lactobacillus, Propionibacterium and Erysipelothrix which are Gram-positive.

Reagents:

A. Crystal violet solution:

Crystal violet … 0.5 gm.

Distilled water to make … 100 ml.

First make a thick solution of crystal violet in absolute alcohol, then make up the volume to 100 ml with distilled water.

B. Gram’s iodine:

Iodine … 1 gm.

Potassium iodide … 2 gm.

Distilled water to make … 100 ml

At first dissolve pot. iodide in 50 ml water. Then add iodine and dissolve in it. Add more water to make the volume 100 ml.

C. Acetone or alcohol

D. Safranin:

Sufranin … 0.5 gm.

Distilled water to make … 100 ml

Mechanisms of Gram’s stain:

The exact mechanism is not understood.

There are two major theories in this respect:

1. Protoplasmic pH:

The hydrogen ion concen­tration of the protoplasm of Gram-positive bacteria (pH 2-3) is higher than that of Gram-negative bacteria (pH 4-5). The iodine treatment makes the cytoplasm further more acidic and serves as a mordant, i.e. iodine combines with the dye and then fixes the dye in bacterial cell.

2. Cell wall permeability:

The Gram-reaction probably depends more on the permeability of bacterial cell wall and cytoplasmic membrane. A dye-iodine complex or lake is formed within the cell after staining with crystal/methyl violet and treatment with iodine. This dye-iodine complex is insoluble in water but moderately soluble and dissociable in alcohol or acetone used as the decolorizing agent.

Under the action of the decolouriser, the dye and iodine complex diffuse freely out of the Gram-negative cell, but not from the Gram-positive cell, presumably because the cell wall of the latter is less permeable. Gram-positive bacteria become Gram-negative when their cell wall is ruptured or damaged or deficient (L-form).

ii. Acid-Fast Stain:

It is extremely difficult to stain mycobacteria with ordinary stain like Gram’s stain. Staining of Mycoba­cterium (tubercle and lepra bacilli) can be done by Acid-fast stain (Ziehl-Neelsen technique) or by auramine-rhodamine stain.

The latter is more sen­sitive than the acid-fast stain but requires fluore­scence microscopy. The acid-fast stain was discovered by Ehrlich and subsequently modified by Ziehl and Neelsen.

Procedure:

Smear preparation:

(a) With the help of a sterile swab stick a portion of sputum is picked up and smeared on a clean slides. From culture smear is prepared in normal saline. In cases of M. leprae, scrapp­ing of tissue from slit skin of lesions, using a scalpel blade, is smeared directly on slide. Air dry the smear.

(b) Smear is fixed by gently heating by passing the slide over a flame several times. Care is taken not to overheat.

Staining method:

(a) Hot and steaming carbol fuchsin solution in a test tube is poured over the smear to cover it. It may need intermittent heating of the reverse side of the slide using a spirit flame. Wait for 5-10 minutes. Be careful not to dry the stain. If required add more stain.

(b) Pour off the stain and wash in tap water.

(c) Decolorize the smear with 20% H₂SO₄ till very faint pink colour persists. In case of M. leprae 4-5%. H₂SO₄ is used. Alternative to 20% H₂SO₄, 3% acid-alcohol (3 ml conc. HCl in 97 ml of absolute alcohol) may be used in special circumstances, e.g. demonstration of Crypto­sporidium oocyst in stool, or to differentiate M. tuberculosis complex (both acid and alcohol fast) from some M.O.T.T. bacillus.

(d) Next counterstain using 2% alkaline methy­lene blue or malachite green (same as Albert I) solution for 1-2 minutes.

(e) Gently wash with tap water and air or blot dry.

Examination:

Scan the slide thoroughly, at least 200 fields, under oil immersion objective with condenser up.

Observation:

(fig. 2.7 C & D)

Acid-fast bacilli appear red in blue background of pus cells and epithelial cells in smear of sputum.

Acid-fast organisms:

1. Mycobacterium-Tubercle and lepra bacilli.

2. Others-Bacterial spores, ascospores of some yeasts, Actinomyces clubs (in animal tissue), some Nocardia species and Cryptosporidium oocysts. The bacterial spores are weakly acid- fast where the decolorizing agent used is 0.5% H₂SO₄.

Principle of acid-fast staining:

Acid-fastness has been attributed to the high content of lipids, fatty acids and higher alcohols, which constitute almost 40% of the dry weight of tubercle bacilli. Mycolic acid (a wax), acid-fast in the free state, is found in all acid-fast bacteria. The integrity of the cell wall is also important for acid-fastness of bacteria.

Reagents:

1. Ziehl-Neelsen’s Carbol fuchsin solution:

Basic fuchsin…. 1 gm.

Phenol (crystalline)…. 5 gm.

Alcohol (95% or absolute)…. 10 ml

Distilled water to make…. 100 ml

Dissolve the dye in the alcohol and add the same to 90 ml 5% phenol solution.

2. Methylene blue (2%)

Saturated solution of methylene blue in alcohol…. 30 ml

KOH, 0.01% in water…. 100 ml

iii. Albert’s Staining:

Albert stain is done to differentially stain the metachromatic granules and cytoplasm of Corynebacterium diphtheriae and diphtheroid bacillus.

Procedure:

(a) Fixation of smear is done by gentle heating over a flame avoiding overheating to char.

(b) Cover the smear with Albert-I staining solu­tion. Keep it for 5 minutes.

(c) Pour off the stain. Do not wash.

(d) Next stain the smear for 2 minutes with Albert-II staining solution (iodine solution).

(e) Pour off the Albert-II solution from slide. Do not wash.

(f) Blot to dry the smear.

(g) Examine the smear under oil immersion lens (x 100) with condenser up.

[Note: Like other methods of bacterial staining, in Albert method, washing is avoided as malachite green is highly soluble in water and stain quality fades].

Staining reagents:

A. Albert I or Albert stain:

1. Toluidine blue …. 0.15 gm.

2. Malachite green …. 0.20 gm.

3. Glacial acetic acid ….1 ml

4. Alcohol (95% ethanol) ….2 ml

5. Distilled water to make …. 100 ml

Dissolve the dyes in alcohol and add to the water and acetic acid, allow the stain to stand for one day and then filter.

B. Albert II or Albert’s iodine solution:

1. Iodine…. 2 gm.

2. Potassium iodide…. 3 gm.

3. Distilled water to make…. 300 ml

Dissolve KI in water and then add Iodine and dissolve in pot. iodide.

Observation:

Body of the bacilli appear green and metachromatic granules blue-black (Fig. 3.1).

Internal quality control of staining:

Quality of staining reagents and procedure is checked every time when new staining solution is prepared (or procured) and at the time of staining, identification of unknown bacteria in culture and clinical material. Bacterial smears with known staining character are subjected for staining. If the results are in expected line, the reagent and the procedural quality is validated.

Example:

(a) Gram stain:

Positive control …. Staphylococcus

Negative control …. E. coli

(b) Acid Fast stain:

Positive control …. Mycobacterium tuberculosis

Negative control …. E. coli

(c) Albert stain:

Positive control …. Corynebacterium diptheriae

Negative control …. None

Some points to note:

1. Morphology by staining is best studied in stationary phase of growth of bacteria.

2. Gram stain cannot stain bacterial spores, which appear as clear ‘halo’ inside the cell cytoplasm.

3. Spores may be stained by cold carbol-fuchsin, followed by decolourisation with 0.5% H₂SO₄.

4. Albert I staining solution imparts green stains to the cytoplasm of all gram +ve and —ve bacteria.

5. An old gram +ve bacterial culture may appear gram -ve or gram variable.

6. Spirochaetes needs D.G.I and metal-shadowed preparations for study of morphology.