BIOCHEMICAL TEST | Bacterial Identification Technique | Microbiology | Vivek Srinivas |#Bacteriology

Summary

This video presentation delves into various biochemical tests for bacterial identification, essential for definitive diagnosis beyond clinical signs. It covers direct microscopic examination, culturing on different media, and a range of biochemical tests including catalase, oxidase, indole, methyl red, Vogus Proskauer, citrate utilization, urease, sugar fermentation, hydrogen sulfide production, and the Triple Sugar Iron (TSI) test. The script also mentions advanced techniques like PCR, DNA sequencing, and phage typing, concluding with the gelatin liquefaction test, providing a comprehensive guide to bacterial identification in laboratories.

Takeaways

• 🔬 The definitive identification of bacteria requires laboratory examination beyond clinical signs and symptoms.
• 📝 Direct microscopic examination and bacterial staining techniques like Gram, acid-fast, negative, spore staining, and motility tests are initial steps in bacterial identification.
• 🌱 Culturing bacteria on different types of media such as differential, selective, enriched, and enrichment broth is essential for further analysis.
• 🧪 Biochemical tests are crucial for identifying the genus and species of bacteria by assessing their enzymatic properties and substrate utilization.
• 🌡 The catalase test differentiates bacteria based on their ability to decompose hydrogen peroxide, producing oxygen bubbles for positive reactions.
• 🟣 The oxidase test detects the presence of the oxidase enzyme, which turns a specific reagent purple in the presence of bacteria.
• 🔴 The indole test identifies bacteria that produce indole as a by-product when metabolizing tryptophan, indicated by a red color change with Kovax reagent.
• 🍬 The methyl red test measures the acidic by-product of glucose fermentation, which changes the indicator to red in a positive test.
• 🍇 The Voges-Proskauer (VP) test detects the production of acetoin from glucose fermentation, resulting in a red color complex with specific reagents.
• 🍋 The citrate utilization test identifies alkaline pH changes due to citrate metabolism, indicated by a color change to Prussian blue.
• 🔄 The IMViC tests (Indole, Methyl Red, Voges-Proskauer, and Citrate) are a set of biochemical tests used to differentiate members of the Enterobacteriaceae family.
• 🧫 The urease test detects bacteria that hydrolyze urea into ammonia and carbon dioxide, causing an alkaline pH shift indicated by a color change to pink.
• 🍯 The sugar fermentation test determines a bacteria's ability to ferment various sugars, producing acid and possibly gas, which can be observed through color change and bubble formation.
• ⚫ The hydrogen sulfide production test detects the formation of black precipitate due to the reaction between hydrogen sulfide and iron salts.
• 🌊 The triple sugar iron (TSI) test evaluates sugar fermentation and hydrogen sulfide production using a medium with three different sugars and an iron salt indicator.
• 🧊 The gelatin liquefaction test detects bacteria that produce gelatinase, an enzyme that breaks down gelatin even at low temperatures, maintaining a liquefied state.

Q & A

What is the primary purpose of biochemical tests in identifying bacteria?

The primary purpose of biochemical tests is to provide definitive identification of bacteria, which is necessary beyond just clinical signs and symptoms, to identify the causative bacterial agent for infection.

Why is laboratory examination essential for bacterial identification?

Laboratory examination is essential because it allows for the collection of correct samples and the application of various tests, such as microscopic examination, culturing, biochemical tests, serological tests, molecular techniques, and phage typing, which are necessary for definitive bacterial identification.

What is the principle behind the catalase test for bacterial identification?

The principle behind the catalase test is that certain bacteria produce the enzyme catalase, which can decompose hydrogen peroxide into water and oxygen. The release of oxygen is observed as bubbles or effervescence, indicating a positive test.

How is the oxidase test performed and what does it indicate?

The oxidase test is performed by placing a bacterial colony on an oxidase reagent-impregnated disc. If a purple color develops, it indicates the presence of the oxidase enzyme in the bacteria, suggesting a positive test.

What is the purpose of the indole production test and how is it conducted?

The indole production test is used to detect the presence of the enzyme tryptophanase, which converts tryptophan into indole. The test is conducted by inoculating a bacterial colony into a broth containing tryptophan and adding Kovax reagent; a red color development indicates a positive test due to indole production.

What does the methyl red test indicate about bacterial metabolism?

The methyl red test indicates whether certain bacteria ferment glucose to produce pyruvic acid, which lowers the pH to acidic levels. The addition of methyl red indicator to the broth changes the color to red in acidic conditions, indicating a positive test.

What is the significance of the Voges-Proskauer (VP) test in bacterial identification?

The VP test is significant as it detects the production of acetylmethylcarbinol (diacetyl), a neutral byproduct of glucose metabolism in certain bacteria. A positive test shows a red color change when specific reagents are added, indicating the presence of this byproduct.

How does the citrate utilization test help in bacterial identification?

The citrate utilization test helps in identifying bacteria that can utilize sodium citrate to produce carbonate and bicarbonate, which raises the pH to alkaline levels. A color change to prussian blue in the presence of bromothymol blue indicator indicates a positive test.

What is the principle of the urease test and what does a positive result indicate?

The principle of the urease test is based on the ability of certain bacteria to produce the enzyme urease, which hydrolyzes urea into ammonia and carbon dioxide, increasing the pH to alkaline levels. A positive result is indicated by a color change to pink due to the presence of phenol red indicator.

What information can be gathered from the sugar fermentation test?

The sugar fermentation test provides information about the ability of bacteria to ferment specific sugars, producing acid as a byproduct and changing the pH to acidic levels. This can help differentiate between bacterial species based on their metabolic capabilities.

How does the triple sugar iron (TSI) test differentiate between different bacterial species?

The TSI test differentiates bacterial species by observing their fermentation patterns of three different sugars (glucose, sucrose, and lactose) and their ability to produce hydrogen sulfide. Different reactions, such as color changes and gas production, indicate the metabolic characteristics of the bacteria.

What does the gelatin liquefaction test measure and what is its significance?

The gelatin liquefaction test measures the ability of certain bacteria to produce the enzyme gelatinase, which breaks down gelatin into amino acids, even at low temperatures. A positive test, where the gelatin remains liquefied at 4 degrees Celsius, indicates the presence of this enzyme.