Transcription
[Music] In this video presentation, we will see about the different biochemical tests for the identification of the bacteria. The diagnosis of the bacterial infection can be done based on the clinical signs and the symptoms of the disease, but it gives only the tentative identification. But for the definitive identification, the laboratory examination is necessary. By the laboratory examination, we can identify the causative bacterial agent for the infection. But for that, the correct sample from the patient has to be collected based on the clinical signs.
First, the collected sample can be subjected for the direct microscopic examination by performing the different bacterial staining techniques like the Gram staining, the acid-fast staining, the negative staining, the spore staining, and the motility test. Next, by culturing the bacteria onto the different types of bacterial culture media like the differential media, the selective media, the enriched media, and the enrichment broth. And next, by the biochemical tests. These tests will be detailed in this video presentation.
Next, by the serological tests like the slide agglutination test, the tube agglutination test, and the microscopic agglutination test. And next, by using molecular techniques like the PCR followed by the DNA sequencing. And lastly, the phage typing, that is, the bacterial identification by using the bacteriophages. These are the laboratory methods and the strategy followed for the bacterial identification.
Only by the microscopic examination and by the culturing technique, the complete identity of the bacteria is not possible. For instance, the bacteria producing the pink colonies on the MacConkey's agar, when subjected to the microscopic examination, will show the Gram-negative bacilli, which may be the E. coli, the Klebsiella, or the Citrobacter. So, the genus and species level bacterial identification is achieved by subjecting the bacterial culture to various biochemical tests and other techniques.
The biochemical tests help in the identification of the bacterial genus and the species, sometimes even the subspecies too. This is achieved by subjecting the bacterial culture to the various biochemical tests. The various bacteria have its inherent property to produce the different types of enzymes and substances. In addition, the various bacteria also has its inherent property to ferment or to utilize the different types of sugars and substrates, yielding the different types of by-products. This can be identified by subjecting the bacterial culture to the various biochemical tests.
First, we see about the catalase test. The principle: certain bacteria produce the enzyme, the catalase. This enzyme, along with the hydrogen peroxide, is capable of decomposing the hydrogen peroxide to water and oxygen. The release of the oxygen is observed as bubbles or effervescence formation. This is the principle behind the catalase test.
The procedure: take a glass slide, place 0.5 ml of the hydrogen peroxide over the slide. Then emulsify the bacterial colony to the hydrogen peroxide. If there are no bubbles or no effervescence observed, it indicates the test is negative. Example: the Streptococcus bacterial organism is negative for the catalase test. In contrast, if the bacterial colony, once emulsified with the hydrogen peroxide, produces bubbles or effervescence, it indicates the test is positive. Example: the Staphylococcus bacterial organism is positive for the catalase test. This is the procedure for the catalase test. This is a photograph showing the positive and the negative test result of the catalase test.
Next, the oxidase test. The principle: certain bacteria produce the enzyme, the oxidase. This enzyme, along with the oxidase reagent consisting of one percent tetramethylparaphenyldiamine dihydrochloride, is capable of turning into the endophenol. This into phenol is a purple color complex. This is the principle behind the oxidase test.
The procedure: take the oxidase disc. This disc is the oxidase reagent impregnated disc. Now, place the bacterial colony over the oxidase disc. If there is no color development observed, it indicates the test is negative. Example: the E. coli bacterial organism is negative for the oxidase test. In contrast, following addition of the bacterial colony over the oxidase disc, it produces the purple color, indicating the test is positive. Example: the Pseudomonas bacterial organism is positive for the oxidase test. This is the procedure for the oxidase test. This is a photograph showing the positive and the negative test result of the oxidase test.
Next, the indole production test or the indole ring test. The principle: certain bacteria produce the enzyme, the tryptophanase. This enzyme, in the presence of tryptophan, is capable of utilizing the tryptophan to yield indole as the by-product. Next, this indole by-product, once mixed with the Kovacs reagent, develops a red color complex. This is observed as the red ring. This is also referred to as the indole ring. This is the principle behind the indole ring test.
The procedure: this test is performed in a test tube with the liquid broth containing tryptophan. Now, take the bacterial colony, inoculate into this broth, incubate for 24 to 48 hours. Following incubation, add the Kovacs reagent along the side. The formation of a red ring indicates the test is positive due to indole production. Example: the E. coli bacterial organism is positive for the indole test. In contrast, following addition of the Kovacs reagent, if there is no red ring formation, it indicates the test is negative for indole production. Example: the Salmonella bacterial organism is negative for the indole test. This is the procedure for the indole test. This is a photograph showing the positive and the negative test result of the indole production or the indole ring test.
Next, the methyl red test, also abbreviated as the MR test. The principle: certain bacteria, in the presence of glucose, ferment the glucose to yield pyruvic acid as the by-product. This changes the pH to acidic from the initial neutral pH. Next, this acidic pH, along with the methyl red indicator, develops the red color. This is the principle behind the methyl red test.
The procedure: this test is performed in the glucose phosphate broth with a neutral pH. Now, take the bacterial colony, inoculate into this broth, incubate for 24 to 48 hours. Following incubation, add the methyl red indicator to it. The development of red color indicates the test is positive due to acidic pH. Example: the E. coli bacterial organism is positive for the MR test. In contrast, following addition of the methyl red indicator, if there is no color change, it indicates the test is negative for the MR test. Example: the Klebsiella pneumoniae bacterial organism is negative for the MR test. This is the procedure for the methyl red test. This is a photograph showing the positive and the negative test result of the methyl red test.
Next, the Voges-Proskauer test, also abbreviated as the VP test. The principle: certain bacteria, in the presence of glucose, ferment the glucose to the acid and then subsequently metabolize to yield a neutral by-product, the acetyl methyl carbonyl, which is an acetoin compound. Next, this acetoin compound, along with the 40% potassium hydroxide and the 5% alpha-naphthol, forms the red color complex, the diacetyl derivative. This is the principle behind the Voges-Proskauer test.
The procedure: this test is performed in the glucose phosphate broth. Now, take the bacterial colony, inoculate into this broth, incubate for 48 hours. Following incubation, add the 5% alpha-naphthol and the 40% potassium hydroxide to it. The development of the red color indicates the test is positive due to diacetyl formation. Example: the Klebsiella pneumoniae bacterial organism is positive for the VP test. In contrast, following addition of the 5% alpha-naphthol and the 40% potassium hydroxide, if there is no color change, it indicates the test is negative for the VP test. Example: the E. coli bacterial organism is negative for the VP test. This is the procedure for the VP test. This is a photograph showing the positive and the negative test result of the VP test.
In general, the VP test is done in conjugation with the MR test. The bacterial organisms showing the MR test positive will give the VP test negative, and the bacterial organisms showing the MR test negative will give the VP test positive. Therefore, in general, these two test results are vice versa for the bacteria.
Next, the citrate utilization test. The principle: certain bacteria, in the presence of sodium citrate, utilize the citrate to yield carbonate and bicarbonate as the by-product. This changes the pH to alkaline from the initial neutral pH. This is the principle behind the citrate utilization test.
The procedure: this test is performed in a test tube with the agar slant prepared with the Simmons citrate agar containing the sodium citrate and the bromothymol blue as the indicator. This bromothymol blue indicator at neutral pH appears as a deep forest green. Now, take the bacterial colony, inoculate into this agar slant, incubate for 24 to 48 hours. Following incubation, if the agar slant turns to Prussian blue, it indicates the test is positive due to the changes in the pH to alkaline because of the yield of the carbonate and the bicarbonate as the by-product. This bromothymol blue indicator at alkaline pH appears as Prussian blue. Example: the Salmonella bacterial organism is positive for the citrate test. In contrast, following incubation, if there is no color change, it indicates the test is negative for the citrate utilization test. Example: the E. coli bacterial organism. This is the procedure for the citrate utilization test. This is a photograph showing the positive and the negative test result of the citrate utilization test.
The IMViC reactions: there are a set of four biochemical tests. They are commonly employed in the identification of the members of the family Enterobacteriaceae. The four biochemical tests are the indole ring test, the methyl red test, the Voges-Proskauer test, and the citrate utilization test. The letter 'I' is only for the rhyming purpose. The E. coli bacteria gives ++-- for the IMViC reactions. The Salmonella bacteria gives -++- for the IMViC reactions. The Klebsiella bacteria gives --++ for the IMViC reactions. So, IMViC reactions are employed in the identification of the members of the family Enterobacteriaceae.
Next, the urease test, also referred as the urea hydrolysis test. The principle: certain bacteria produce the enzyme, the urease. This enzyme, in the presence of urea, is capable of hydrolyzing the urea to yield ammonia and carbon dioxide as the by-product. This by-product changes the pH to alkaline from the initial neutral pH. This is the principle behind the urease test.
The procedure: this test is performed in a test tube with the agar slant containing the urea and the phenol red as the indicator. This phenol red indicator at neutral pH appears as orange. Now, take the bacterial colony, inoculate into this agar slant, incubate for 24 to 48 hours. Following incubation, if the agar slant turns to pink, it indicates the test is positive due to the changes in the pH to alkaline because of the yield of the ammonia and the carbon dioxide as the by-product. This phenol red indicator at alkaline pH appears as pink. Example: the Staphylococcus bacterial organism is positive for the urease test. In contrast, following incubation, if there is no color change, it indicates the test is negative for the urease test. Example: the E. coli bacterial organism is negative for the urease test. This is the procedure for the urease test. This is a photograph showing the positive and the negative test result of the urease test.
Next, the sugar fermentation test. Each bacterium has the ability to ferment certain sugars and not ferment certain sugars. To find out the sugar fermentation of the particular bacteria, this test is done. The principle: the bacteria, in the presence of certain sugar, the sugar may be glucose, or sucrose, or lactose, or maltose, or galactose, or mannose, or any other sugars. This bacteria, in the presence of certain sugar, ferments it, yielding acid as the by-product. This changes the pH to acidic from the initial neutral pH. Next, this acid, along with the Andrade's indicator, changes to the pink color due to acidic pH. Some bacteria may also release gases along with the acids. This is observed as bubble formation. This is the principle behind the sugar fermentation test.
The procedure: this test is performed in a broth containing sugar with a Durham's tube inside it. The sugar may be glucose, or sucrose, or lactose, or maltose, or galactose, or mannose, or any other sugars that can be added to the broth. For instance, this broth is added with lactose, thereby to check the bacteria for lactose fermentation. Now, take the bacterial colony, inoculate into this broth, incubate for 24 to 48 hours. Following incubation, add the Andrade's indicator to it. The development of the pink color indicates the test is positive for lactose fermentation due to acid production. Sometimes, bubble formation may be observed in the Durham's tube, indicating gas production. Example: the E. coli bacterial organism is positive for lactose fermentation. In contrast, following addition of the Andrade's indicator, if there is no color change, it indicates the test is negative for lactose fermentation. Example: the Salmonella bacterial organism is negative for lactose fermentation. This is the procedure for the sugar fermentation test.
Next, the hydrogen sulfide production test. The principle: certain bacteria produce a substance like hydrogen sulfide. This hydrogen sulfide, along with iron salts like ferrous sulfate and ferric ammonium salt, forms ferrous sulfide as the end product. This is observed as a black precipitate. This is the principle behind the hydrogen sulfide production test.
Next, the triple sugar iron test, also referred as the TSI test. The procedure: this test is performed in a test tube with the agar slant prepared with the TSI agar containing the three sugars: glucose, sucrose, and lactose, to check the bacteria for sugar fermentation for these three sugars. And also contains ferrous sulfate salt to check the bacteria for hydrogen sulfide production. And contains the indicator, the phenol red. This phenol red indicator at neutral pH appears as orange. Generally, the top slanting portion of the agar is referred as the slant, and the bottom portion of the agar is referred as the butt. The three sugars present in the TSI agar are in different concentrations, such as glucose 0.1%, sucrose 1%, and lactose 1%. This is about the TSI agar.
Now, take the bacterial colony, inoculate into this agar slant, incubate for 24 to 48 hours. Following incubation, the agar slant may turn to this type of reaction. This type of reaction is observed in the E. coli, that is, the yellow slant due to acidic pH at the slant portion, and the yellow butt due to acidic pH at the butt portion, and the bubble cracks and the displacement of the agar due to gas production. Since the E. coli ferments all the three sugars present in the media, so a large amount of acids are produced, which turns the phenol red indicator to yellow both at the butt portion and at the slant portion. This type of reaction is observed in the E. coli.
Following incubation, the agar slant may turn to this type of reaction. This type of reaction is observed in the Salmonella, that is, the red slant due to alkaline pH at the slant portion, and the yellow butt due to acidic pH at the butt portion. And also, we can observe the presence of the black precipitate due to hydrogen sulfide production. Remember, the butt portion comparatively contains more glucose compared to the slant portion. Since the Salmonella ferments only glucose present in the media, a small amount of acid is produced, which turns the phenol red indicator to yellow only at the butt portion. This type of reaction is observed in the Salmonella.
These are some of the reactions observed in the TSI agar, starting from the left: the uninoculated TSI agar, the middle: the E. coli inoculated TSI agar, and the right: the Salmonella inoculated TSI agar. This is a photograph showing the reactions observed in the TSI agar, the left: the uninoculated TSI agar, the middle: the E. coli inoculated TSI agar, and the right: the Salmonella inoculated TSI agar. Therefore, the E. coli ferments all the three sugars present in the media and turns the phenol red indicator to yellow both the butt portion and the slant portion. But the Salmonella ferments only glucose present in the media and turns the phenol red indicator to yellow only the butt portion.
Next, the gelatin liquefaction test, also referred as the gelatin hydrolysis test. In this experiment, the gelatin will be used. Before that, we will see about the property of the gelatin. The gelatin at high temperature, that is, at temperature above 25 degrees Celsius, it liquefies. In contrast, the gelatin at low temperature, that is, at temperature below 15 degrees Celsius, it solidifies. This is the property of the gelatin. Now, we will see about the principle of this test. Certain bacteria produce the enzyme, the gelatinase. This is a proteolytic enzyme. This enzyme, in the presence of gelatin, is capable of breaking down the gelatin into individual amino acids as the end product. This liquefies the gelatin and remains liquefied even at 4 degrees Celsius, that is, remains liquefied even at the chilling temperature. This is the principle behind the gelatin liquefaction test.
The procedure: this test is performed in a test tube prepared with the nutrient gelatin medium. Now, take the bacterial colony, inoculate into this gelatin medium, incubate for 48 hours. Following incubation, if the medium remains liquefied even at 4 degrees Celsius, it indicates the test is positive due to the liquefaction of the gelatin. Example: the Staphylococcus aureus bacterial organism is positive for the gelatin liquefaction test. In contrast, following incubation, if the medium remains solidified at 4 degrees Celsius, it indicates the test is negative due to the gelatin is not hydrolyzed by the bacteria. Example: the E. coli bacterial organism is negative for the gelatin liquefaction test. This is the procedure for the gelatin liquefaction test. This is a photograph showing the positive and the negative test result of the gelatin liquefaction test.
The schematic illustration of the different biochemical tests is available as a downloadable link in the below YouTube description. In the next video presentation, we will see about the antimicrobial susceptibility test. Stay tuned to this YouTube channel. Hope the lecture is informative and useful. Thank you.