Observing and Examining Micro-Organisms (Microbes)

After reading this article you will learn about observing and examining micro-organisms.

Observation of Microbes in Hanging Drop:

This method is used for the study of motility, germination or fission of micro-organisms which is not possible when they are killed and fixed in stained preparations.

For this observation the following are needed:

1. A depression slide with a circular concavity in the centre,

2. A cover for glass to fit on the concavity,

3. Distilled water if culture is in solid medium,

4. Transferring needle with a loop, and

5. The culture of the microbe to be examined.

The periphery of the concavity on the depression slide is smeared with vaseline. A loop- full of bacterial culture or spore suspension or any other microbial suspension is placed in the centre of the cover glass if it is a liquid culture.

In case of solid culture it is mixed with a drop of distilled water and placed in the centre of the cover glass. The cover glass with the drop of culture is inverted over the concavity so that the drop is in the centre and the edge of cover glass fits closely to the slide, the vaseline acting as a adhesive. Observations of the hanging drop are done now under the microscope.

Procedure for Examining Microbes:

Temporary mounts of fungous mycelium or its spores or protozoa can be made for observation in water covered by cover-glass. The edges may be sealed with wax to prevent quick drying. Glycering-water may also be used as a mounting medium. In case the material is hyaline it may be stained with suitable stain, for instance, for fungi cotton blue may be used and mounted in lacto phenol.

Bacteria are too small and so need to be killed and fixed on a clean slide and then stained for observation under oil immersion. For this purpose a small amount of culture is transferred to a drop of distilled water placed on a clean slide and a thin smear prepared with the aid of the transferring needle.

The smear is allowed to dry in air and fixed by passing the slide with film side upwards into flame two to three times. It is now killed and fixed. It is then stained with one of the aniline dyes (methylene blue, gentian violet, methyl violet etc.).

Excess stain may be removed with water and dried for observation under oil immersion lens. The bacteria may also be killed and fixed with methyl alcohol or any other suitable fixative. The staining of such preparation must, however, be done after washing it in water.

Stains and Staining:

Stains are dyes or reagents used for colouring bacteria or other micro-organisms in order to observe them more clearly under microscope and do differentiate them (e.g., gram, acid fast etc.), and to inhibit the growth of some so that others uninhibited may be studied (e.g. Brilliant green inhibits Escherichia coli in a medium but not Salmonella species, gentian violet inhibits most gram positive, fast growing bacteria, Rose-bengal retards the spread of fungi).

The dyes are organic compounds, composed of benzene with a colour group (chromophore) which is responsible for a specific colour production when it associates with the micro-organism to be stained through another moiety of the dye known as auxochrome.

The two types of dyes viz. the basic and acid dyes are named after the basic (NH or N (CH,)₂) or acid (SO₃H and COOH) reaction of the auxochrome. In staining, in order to render the colour fast or increase the retention ability of the colour a mordant is used such as Lugol’s solution (iodine in potassium iodide, ferrous sulphate, tannic acid etc.)

Stains are of Various Kinds:

(i) Direct or general stains. Aniline dyes are able to stain bacteria directly. The spores of bacteria (Bacillus spp.) and some bacteria which have waxy coating on cell wall (Mycobacterium spp.) fail to stain with these stains.

(ii) The indirect stains are dyes which only stain the background like nigrosin or India ink used either for observing mucilaginous covering enveloping bacteria (capsules) or certain spores of fungi or cells of unicellular animals.

(iii) Selective stains are used for special purpose, to stain particular parts of the organisms such as spores, metachromatic granules, flagella, nuclei etc.

(iv) Differential stains are those which enable one to differentiate two different groups of bacteria in a mixture, for instance, Gram-positive and Gram-negative.

In general, stains are prepared from saturated and filtered alcoholic or aqueous solutions. Some of the more commonly used stains in microbial studies are, cotton blue, safranine, carbol fuchsin and aniline dyes.

Gram Staining:

Gram staining is a very useful and commonly used differential stain. While defining species its reaction to this stain has to be included.

The procedure for staining is as following:

1. A thin film of test bacterium (smear) is fixed on a clean slide as described elsewhere.

2. The smear is stained first with any aniline dye such as gentian violet, methyl violet, crystal violet etc. in distilled water, by immersing the smeared portion of the slide in the stain for about 2 minutes.

3. The excess stain is removed by washing in running tap water and the slide immersed in a mordant (Lugol’s solution containing 1 gm. of iodine and 2 gm. of potassium iodide in 300 ml. of distilled water with alkaline reaction) for about 2 minutes in order to fix the colour.

4. The slide is now washed with a 95% alcohol till no more colour is removed from the slide. If bacteria are completely de-stained (lose colour) in this step, they would be Gram-negative. However, if they retain the colour despite this treatment they would be considered Gram-positive.

5. Finally the slide is immersed in 0.2% aqueous solution of safranine so that the Gram-negative bacteria which have lost the colour during de-staining may show up as pink objects under microscope.

Great care should be exercised while using this staining method because Gram-positive bacteria may became Gram-negative with age. It is, therefore, advisable to use young cultures (18-24 hours old). Most cocci are Gram-positive. The genus Neisseria (a diplo-coccus) for instance is Gram-negative.

The Gram-negativeness of a bacterium is said to be due to the presence of large amounts of certain proteins and lipids with poly-saccharides in the cell wall absentin cell wall of Gram-positive bacterium.

Further, when cell wall is removed from Gram-positive bacterium, the stained protoplasm loses its colour in treatment with a destaining agent suggesting that Gram-positiveness is due to special chemical composition of the cell wall.

In Gram-negative bacteria the high lipid content of cell wall is said to permit the action of destaining agent on the stained protoplasm. This component being absents in the cell wall of Gram-positive bacteria the entry of alcohol or any destaining agent through the cell wall for action on the stained protoplasm is not rendered possible.

Spore Staining:

After preparing a smear of the spore former, it is air-dried and fixed with gentle heat. Pouring excess carbol fuchsin on the smear with a dropper the non-smeared side of the slide is heated under low flame for three minutes so that the slow vaporization of the reagent occurs.

The slide thereafter is washed in 5% acetic acid until the smear appears light pink. It is then counter-stained with Loffler’s methylene blue for 3 minutes and excess stain washed and air dried for observation or moisture removed with a blotting paper. On examination the pink spores within blue vegetative cells can be observed under the microscope.

Capsule Staining (Welch’s method):

The smear of bacteria is air-dried and fixed with gentle heat. The smear is then covered with glacial acetic acid for a few seconds and drained. The acetic acid is later washed with gentian violet in aqueous solution ultimately leaving the dye for about 3 minutes. The slide is finally washed in 2% salt solution and examined. The bacterial cells are surrounded by pale violet capsules.

Gin’s Method of Staining Capsule:

A drop of 1: 1 solution of India ink in sterilised distilled water is mixed with a loopful of the bacterial culture on a clean slide at one edge. With the edge of another slide a thin smear is prepared, air-dried and fixed either with gentle heat or methyl alcohol. The smear is now stained with gentian violet for 1-2 minutes (carbol fuchsin may also be used).

The excess stain is washed in water, dried and examined. The cells are seen violet or pink depending upon whether gentian violet or carbol fuchsin is used, surrounded by an unstained capsule region, its margin made distinct by India ink.

Flagella Staining:

The test 18 hour old bacterial culture is transferred to a small amount of water (2 ml) in a test tube and incubated at 37°C for about 15 minutes. A loopful of this is used for preparation of a thin film on non-greasy clean slide. The smear is the air-dried and fixed as usual.

Staining Method:

1. Loffler’s:

The filtered mordant with the following composition is allowed to act for about 1 minute on the smear with gentle heat. The mordant should not be permitted to boil. The slide is then washed in water. Filtered 5% alkaline aqueous solution of gentian violet or carbol fuchsin is poured on the smear and allowed to act for about 2 minutes, then washed and air-dried for observation.

Mordant:

2% aqueous tannic acid – 10 parts

Saturated aqueous solution of ferrous sulphate – 7parts

Saturated alcoholic fuchsin solution – 1part

2. Casares-Gil’s:

Filtered one part of the mordant of the following composition with two parts of distilled water is poured on the smear and allowed to act for about 2 minutes. When precipitate and metallic sheen begin to appear, the slide is washed in distilled water and covered with carbol fuchsin for about 2 minutes, then washed in distilled water and dried in air for examination.

Mordant:

Tannic acid – 10 gm.

Aluminium chloride – 18 gm.

Zinc chloride – 10 gm.

Rosaniline hydrochloride – 1.5 gm.

60% alcohol – 40 gm.

Acid-fast Bacteria:

A stained bacterium that does not decolorise on treatment with dilute acid is said to be acid fast. However an acid-fast bacterium stained after treatment with a defating agent loses the stain with acid treatment showing that the acid fast nature is due to presence of high content of lipids.

Staining by Ziehl Neelsen’s Method:

The smear of the culture is prepared on a slide. It is covered by carbol fuchsin (10 ml of saturated alcoholic solution of basic fuchsin in 90 ml of 5% aqueous phenol solution) and steamed on a low flame for 5 minutes without boiling, adding fresh amounts of stain to prevent drying of the slide.

It is then washed in water, and treated with decolorizing agent (3 ml hydrochloric acid in 97 ml 95% alcohol) and steamed for 10-30 minutes with methylene blue, washed and dried for examination. Acid fast bacteria are bright pink against blue back­ground.

Burries India Ink method for Staining Spirochaetes:

A loopful of the spirochaete suspension is mixed with equal amount o India ink solution (1:10 India ink solution in water that is sterilised for 15 minutes in steam) on a clean slide and a thin smear is prepared. It is then dried and examined under dark field illumination. The spirochaetes are seen as white bodies against dark field.

Staining Protozoa:

A film of the protozoa is prepared on a slide and fixed by first treating with warm (60°C) solution containing 2 parts of saturated mercuric chloride, 1 part of absolute alcohol and glacial acetic acid (5 ml of glacial acetic to 100 ml of the mixture).

The slide is then rinsed in 50% alcohol, 70% alcohol iodine, 70% alcohol and 50% alcohol series for two minutes in each. Rinsing in water, it is placed for 10 minutes in a mordant for 2-4% iron- alum warmed to 30°C.

The slide is now washed in running water for 5 minutes and stained for about 30 minutes in 0.5%. Heidenhain’s ion heamatoxylin (10% solution in absolute ethyl alcohol and a portion diluted to 0.5% strength, the portion depending upon the requirement) at 30°C.

The slide is later washed for 5 minutes in running water and dipped in 2-4% iron-alum to affect a degree of decolorisation that would render nuclei visible. This is done by examination of the preparation under microscope after frequent intervals of treatment.

The slide is treated again in running water for 5 minutes and passed through alcohol and xylol series given below along with periods of treatment in each and mounted in Canada balsam.

50% alcohol – 2 minutes

70% alcohol – do

85% alcohol – do

95% alcohol – 5 minutes

Absolute alcohol – do

Absolute alcohol plus xylol (1: 1) – do

Xylol – do

Staining of Fungi:

Fungi which are hyaline may be observed more clearly by staining in lacto-phenol cotton blue and mounting in lacto phenol.

Lacto phenol:

1. Phenol(liquid) – 500 ml

2. Lactic acid – 500 ml

3. Glycerol – 1000 ml

4. Distilled water – 500 ml

Cotton blue:

1. Phonol – 50 gms

2. Glycerol – 50 ml

3. Lactic acid – 50 ml

4. Distilled water – 50 ml

5. Cotton blue – 2.5 gms