COURSE MLSC 417 LECTURE TOPIC: STAINING METHODS IN BACTERIOLOGY

Introduction Stained preparations are needed to examine micro-organisms microscopically in order to study their morphology and observe their cellular constituents. Smears (or tissue sections) are made and stained by any one of the required staining methods. Smears can be made from liquid or solid cultures or from the clinical specimen..

The staining result is dependent on the quality of the smear. From the liquid culture, smears should be evenly spread on the slide and allow to dry in air. Smears from solid culture should not be thick

Types of staining Indirect staining: This is when an organism is stained only in the presence of a mordant. e.g. Gram's stain. Direct Staining: This a simple one-step staining procedure in which the presence and morphology of bacteria are demonstrated. Some of the stains used for this technique are also used as counterstains in some other staining procedures. e.g.. Methylene blue

.Negative staining: This is when the organism remains unstained against a stained background. This is one of the few methods where acid stains such as nigrosin, are used.   Metachromatic staining: In this staining method the organism or part of the organism is stained a different shade of colour from that of the stain. Commonly used staining methods   In Bacteriology, staining methods are divided into three categories

:. Simple stains: This makes use of the direct staining method : Simple stains: This makes use of the direct staining method. Differential stains: This staining method divides bacteria into two groups Special stains: These are specialised staining methods to demonstrate certain bacterial components, e.g. spore.

Simple staining methods Simple staining methods are used to demonstrate the presence of and the morphology of bacteria and cells. They employ simple stains most of which are also used as counterstains. Few of these methods may contain a mordant e.g. phenol in carbol thionin.

Methylene blue stains Neutral methylene blue, Loeffler's alkaline methylene blue and Polychrome methylene blue solutions are all used in like manner and with almost similar results. The polychrome methylene blue is particularly useful for the identification of anthrax bacilli by Mcfadyean reaction

Staining procedure. Stain the heat-fixed smear for 1 minute Staining procedure Stain the heat-fixed smear for 1 minute. Wash with water. Blot carefully and dry. Results Bacteria and cells:

1. Differential staining methods 1. Differential staining methods Gram's stain In 1884, Christian Gram, a Danish bacteriologist, described this staining method which is the most important stain in routine bacteriology. It divides bacteria into two groups - the Gram positive and Gram negative bacteria.

The Gram positive organism must have an intact cell wall The Gram positive organism must have an intact cell wall. A damaged cell wall inevitably results in Gram negative reaction. This shows the importance of cell wall in Gram stain reaction. The Gram's stain reaction is based on the ability of the organism to resist decolourisation with acetone, alcohol or aniline oil after the initial staining with one of the rosaniline basic dyes and then treating with a mordant. The rosaniline dyes commonly used are crystal violet, methyl violet and gentian violet. Iodine is the mordant used.

A counterstain, contrasting in colour with the primary stain, is used to stain Gram negative bacteria which are decolourised. Gram positive bacteria: In a Gram stained smear Gram positive bacteria which retain the primary stain, appear violet or blue-black or deep purple in colour. The Gram positive reaction is due to the presence of techoic acid in the cell wall. The teichoic acid forms a mesh which prevents the primary stain-mordant complex from being washed away by the decolouring agent.

Gram negative bacteria: These appear red Gram negative bacteria: These appear red. Gram negative bacteria have a high lipid content which dissolves in the decolourising agent. This allows the primary stain-mordant complex to be washed off. o The red counterstain makes the decolourised Gram negative bacteria visible in a contrast colour.

Solutions. Crystal Violet: 0. 5 to 1% in distilled water Solutions Crystal Violet: 0.5 to 1% in distilled water. Lugol's iodine: 10g Iodine 20g Potassium iodide 1000mll Distilled water Dissolve the potassium iodide in about 50 ml of the distilled water, add iodine. Dissolve the iodine by shaking and make up the volume to 1000 ml.

Decolouriser: Solution: Absolute ethyl alcohol or acetone or acetone and alcohol mixture (1:1) Counterstain Aqueous solution of neutral red or safranin 0.5%, or dilute carbol fuchsin. (1:10 dilution of strong carbol fuchin in distilled water)

Procedure Make a smear, allow to dry and then fix with a gentle heat by passing the slide 2 or 3 times over a bunsen flame or placing the slide on a slide warmer. Stain with crystal violet for 1 minute. Wash with tap water. Apply Lugol's iodine and leave for 1 minute. Wash with tap water.

Decolourise with acetone or alcohol until no more colour appears to ooze out of the smear (about 1-2 seconds for acetone and 1-2 minutes for alcohol and 10 seconds for acetone/alcohol mixture) Wash immediately with tap water. Counterstain with neutral red or safranin or dilute carbol fuchsin for 1 minute. Wash with tap water. Blot dry with a blotting or filter paper, and dry.

Examine microscopically using x100 objective. Results. blue/black Examine microscopically using x100 objective. Results blue/black Gram positive bacteria Gram negative bacteria red

(Zehiel-Nelseen method) Acid-fast stain   (Zehiel-Nelseen method) Acid-fast stain Acid-fast staining is another example of a differential stain used in Bacteriology. It divides bacteria into two groups, acid fast-and non acid fast. Members of the genus Mycobacterium are acid-fast in nature. Like the Gram reaction, the acid-fastness is also called cell wall dependent. Mycobacteria have a high lipid content, especially mycolic acid, in their cell wall. The ordinary aniline dye solutions cannot penetrate the mycobacterial cell

wall. Strong staining solutions containing phenol preferably with application of heat, are used for staining them. Once stained, they resist decolourisation with mineral acid. Therefore they are called acid-fast bacilli (AFB). The degree of acid-fastness varies with species. Mycobacterium tuberculosis can resist decolourization with 20% sulphuric acid, or 3% hydrochloric acid in 95% ethanol. Hence they are also called acid-alcohol-fast (AAFB). M.leprae resist 5% sulphuric acid while Nocardia species are acid-fast only with 1% sulphuric acid.

Ziehl-Neelsen (ZN) method. Solutions. Strong carbol fuchsin Ziehl-Neelsen (ZN) method Solutions Strong carbol fuchsin Powdered basic fuchsin 5g Phenol 25g Alcohol 95% 50ml Distilled Water 50ml Dissolve the fuchsin in the solution of phenol with a little water over a boiling water bath. Add the alcohol and mix. Add the rest of the water.

20% sulphuric acid (H2SO4) or 3% acid alcohol (i 20% sulphuric acid (H2SO4) or 3% acid alcohol (i.e 3% hydrochloric acid in 95% alcohol). 0.5% methylene blue or 0.5% malachite green. Procedure Make and fix smear by heat Flood the slide with the carbol fuchsin solution and heat gently until steam rises. Do not allow the stain to boil. Stain for 5-10 minutes and reheat. Leave for another 10 minutes. Wash with tap water.

Decolourise with 20% sulphuric acid or 3% acid alcohol for about 5 minutes. (with 20% H2SO4, until the film becomes yellowish). Wash well in water. Counterstain with 0.5% methylene blue or malachite green for 30 seconds. Wash well with tap water. Blot , dry and examine under oil immersion laws. Results : red Acid-alcohol fast bacteria Background and other bacteria :blue or green.

Kinyoun's method This is a radical modification of the Ziehl-Neelsen's method in which no heating is required.

Auramine phenol fluorescent stain Auramine phenol fluorescent stain This is a fluorescent staining method for the detection of acid fast bacilli. Solutions 1 Auramine phenol 2 3% Acid alcohol 3 0.1% Potassium permanganate Result Acid fast bacilli: Bright luminous rods against dark bachground

Special staining methods   Staining of metachromatic granules Certain bacteria possess granules which are seen in the cytoplasm. The presence of these granules help in the identification of such bacteria. e.g. Corynebacterium species. Special stains such as Albert's, Neisser's and Pugh's stains are used to demonstrate these granules Solutions Albert’s 1 Albert’s 2 Result: Granules: bluish black; Organism: green

Special staining methods   Staining of metachromatic granules Certain bacteria possess granules which are seen in the cytoplasm. The presence of these granules help in the identification of such bacteria. e.g. Corynebacterium species. Special stains such as Albert's, Neisser's and Pugh's stains are used to demonstrate these granules

Staining of capsules Capsules are not stained by ordinary stains due to their low affinity for stains. They are demonstrated by either negative staining or one of the direct staining methods such as the Hiss method.   1. Nigrosin (or India ink) negative staining 2.Nigrosine-methylene blue

Staining of spores The spore wall is resistant to stains but spores can be demonstrated with the application of heat to the preparation.   Malachite green stain for spores 5% malachite green 0.5% safranin

  Staining of bacterial cell wall The composition of cell wall differs from bacteria to bacteria, therefore there are variations in the length of time for the stain and cell wall to interact. The staining method uses tannic acid as mordant and Congo red as a selective decolouriser while crystal violet stains the cell wall, leaving the cytoplasm unstained.

Staining methods for flagella Young cultures are used to demonstrate flagella. Chemically clean slides must be used and the smear made with proper care. All the methods are of silver impregnation techniques.   Fontana's silver method for flagella. 1. 1% Osmic acid 2. Fontana's mordant 10% Tannic acid 10 ml Saturated potassium alum 10 ml

10% Ferric chloride. 1 ml. Mix and stand for 3 minutes before use. The 10% Ferric chloride 1 ml. Mix and stand for 3 minutes before use. The solution does not keep. 3. Fontana's silver solution Dissolve 5g silver nitrate in 100 ml distilled water. Dilute 5 ml of concentrated ammonia with 45 ml distilled water. Add 35 ml of the diluted ammonia solution to 90 ml of silver nitrate.

Continue to add drop by drop until the Continue to add drop by drop until the precipitate which is formed is redissolved. Continue to add more until a faint opalescence is seen. Procedure 1 .Place one drop of 1% osmic acid towards one end of a very clean slide. 2 .With a loop, gently transfer one drop of the water of condensation of an 18 hour old agar slope culture to the drop of Osmic acid and mix gently. Allow the drop to run the whole length of the slide.

3. Allow to dry in air. 4. Add Fontana’s mordant for 3 minutes. 5 3. Allow to dry in air. 4. Add Fontana’s mordant for 3 minutes. 5. Wash in distilled water. 6. Heat Fontana's silver solution in a test tube and flood the slide. Leave for 2 minutes. 7. Wash in distilled water. 9. Dry in air and mount immediately. Results: Organisms : black Flagella : light brown.

Staining method for spirochaetes The usual and easier way of examining spirochaetes is with the darkfield microscope. They do not stain well with aniline dyes and because the spirochaetes spirals are so thin, they are not easily seen by most other staining methods. However, they have the capacity to reduce silver nitrate to metallic silver, thus the demonstration of spirochaetes in smears can be achieved by the silver impregnation method.   .

Fontana's method for spirochaetes Solutions: 1. Fontana's fixative Fontana's method for spirochaetes Solutions: 1. Fontana's fixative Glacial acetic acid 1 ml Formalin 2 ml Distilled water 100 ml 2. Absolute alcohol 3 Fontana's mordant: refer to Fontana’s silver method for flagella 4.Fontana's silver solution: refer to Fontana’s silver method for flagella

Procedure 1. Make a thin smear 2 Procedure 1. Make a thin smear 2. Treat smear with Fontana's fixative 3 times for 30 seconds each. 3. Wash off the fixative with absolute alcohol and cover with absolute alcohol for 3 minutes. 4. Drain off the alcohol and carefully burn off the remainder until the film is dry. 5. Apply Fontana's mordant, heat until steam rises and leave for 30 seconds. 6. Wash well in distilled water and dry the slide.

7. Apply the Fontana's silver solution, heat until steam rises and leave for about ½ minute (the film turns brown in colour). 8. Wash in distilled water, dry and mount in neutral synthetic medium.   Results Spirochaetes : brownish black Background : brownish yellow.