Introduction
Bacteria in nature are found as mixed populations. To study a particular species, it must be isolated and identified. In clinical, industrial and research laboratories, identification of unknown bacteria is an essential task. Bacteria are identified using morphology, staining reactions, physiological properties, biochemical tests and genetic features. Staining techniques and biochemical reactions are two major tools used for routine identification.
Staining Techniques for Bacterial Identification
Staining helps to visualise bacteria, study their morphology and differentiate them based on structural features. Stains contain a chromophore (colour-giving group) and an auxochrome (binding group).
1. Simple Staining
In simple staining, a single basic dye such as methylene blue, crystal violet or safranin is used. The positively charged dye binds to the negatively charged bacterial surface. This method shows the size, shape and arrangement of cells.
2. Negative Staining
Negative stains such as eosin or nigrosin are acidic and carry a negative charge. They do not enter bacterial cells but stain the background. Bacteria appear colourless against a dark background. This method does not require heat fixation and preserves the natural shape of cells. It is useful for studying delicate organisms like spirochetes and for demonstrating capsules.
3. Gram Staining
Gram staining, developed by Christian Gram (1884), is the most widely used differential stain. It divides bacteria into:
- Gram-positive: retain crystal violet and appear purple
- Gram-negative: lose crystal violet, take safranin and appear pink
The staining procedure includes:
- Primary stain – crystal violet
- Mordant – Gram’s iodine, forms CV–I complex
- Decolorizer – alcohol/acetone
- Counterstain – safranin
Gram-positive bacteria retain the stain due to thick peptidoglycan and dehydration by alcohol. Gram-negative bacteria lose the stain because alcohol dissolves outer membrane lipids and increases cell wall permeability.
4. Acid-Fast Staining
Used to identify Mycobacterium and some actinomycetes that contain waxy mycolic acids in their cell wall. Ordinary stains cannot penetrate.
Ziel-Neelsen method:
- Primary stain – carbol fuchsin with heating
- Decolorizer – acid alcohol
- Counterstain – methylene blue
Acid-fast bacteria appear bright pink; non–acid-fast bacteria appear blue.
5. Spore Staining
Used to detect bacterial spores (e.g., Bacillus, Clostridium). Spores resist staining and require heating.
Method: Malachite green stains spores; safranin stains vegetative cells red. Spores appear green, vegetative cells red.
6. Capsule Staining
Capsules are non-ionic and do not bind stains easily.
Two approaches are used:
- Positive staining: crystal violet + copper sulfate; capsule appears light violet
- Negative staining: background stained with nigrosin; capsule appears clear
7. Other Staining Techniques
- Flagella staining: shows presence and arrangement of flagella
- Inclusion staining: detects intracellular storage bodies (glycogen, polyphosphates, etc.)
Biochemical Reactions for Identification
Biochemical tests identify bacteria based on enzyme activity and metabolic reactions. These tests reveal how a microorganism uses nutrients and produces end products.
1. Sugar Fermentation Test
Different sugars are tested in media containing indicators and Durham tubes. Acid production changes media colour (pink/red). Gas collects in Durham tubes.
2. Litmus Milk Reaction
Litmus milk serves as a differential medium. Bacteria metabolise milk components showing reactions such as:
- Lactose fermentation
- Gas production
- Litmus reduction
- Curd formation
- Proteolysis
- Alkaline reaction
3. Indole Production Test
Bacteria with tryptophanase convert tryptophan to indole. After incubation in peptone water, Kovac’s reagent produces a red ring indicating a positive result.
4. Methyl Red (MR) Test
Detects stable acid production from glucose fermentation. A red colour after adding methyl red indicates a positive test.
5. Voges–Proskauer (VP) Test
Detects acetoin production. Addition of KOH and alpha-naphthol produces a red or pink colour in positive organisms.
6. Citrate Utilization Test
Determines ability to use citrate as sole carbon source. Growth and turbidity in Koser’s citrate medium indicates a positive result. MR, VP, indole and citrate tests together form the IMViC tests for enteric bacteria.
7. Nitrate Reduction Test
Detects nitrate reductase enzyme. After incubation, the addition of sulphanilic acid and alpha-naphthylamine produces a red colour if nitrite is present.
8. Hydrogen Sulphide (H2S) Production
Bacteria degrading sulphur-containing amino acids produce H2S, which reacts with lead acetate or iron salts in media, forming black or brown precipitates.
9. Potassium Cyanide (KCN) Test
Tests ability of bacteria to grow in presence of KCN. Growth (turbidity) indicates a positive result.
10. Catalase Test
Detects catalase enzyme which breaks down hydrogen peroxide. Bubbles on adding 10% H2O2 indicate a positive reaction.
11. Urease Test
Detects urease enzyme. Hydrolysis of urea produces ammonia, turning phenol red indicator to deep pink.
12. Oxidase Test
Oxidase-positive organisms oxidize the reagent (tetramethyl-p-phenylenediamine dihydrochloride) to deep purple within seconds.
Key Points
- Staining techniques help differentiate bacteria by structural and chemical properties.
- Gram, acid-fast, spore and capsule stains are essential for routine laboratory diagnosis.
- Biochemical tests identify metabolic and enzymatic characteristics unique to different bacteria.
- IMViC tests help classify Gram-negative enteric bacteria.
- Combining morphology, staining and biochemical data ensures accurate bacterial identification.
Detailed Notes:
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