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Micro Lab Exam 2 - study guide
Terms in this set (26)
Principles and Approaches of bacterial Identification
a) Importance of working with pure culture
b) Morphological characteristics: Gram reaction, cell shape (rods/bacilli, cocci, etc.), and cell arrangement (chains, pairs, tetrads, etc).
c) Cultural and physiological characteristics: motility, fermentation abilities, oxygen requirements, specific enzyme production, etc.
d) Closely related bacteria will share a lot of characteristics due to a very similar genetic arrangement. On the other hand there is a great level of variability among bacterial strains/subspecies that belong to the same species.
ingredients designed to elicit a specific biochemical or physiological response and allow us to differentiate groups of organisms.
components that inhibit the growth of unwanted bacteria and select for/isolate the specific type of bacteria.
Carbohydrate Fermentation Test: Ingredients
One sugar in the broth medium (Glucose, Lactose, Manitol, etc.)
Phenol Red as the pH indicator - red is neutral, yellow indicates acid production
Durham tube - inverted to trap any gas produced
Gelatin in a nutrient broth
Carbohydrate Fermentation Test
This test indicates the microorganism's ability to ferment a particular sugar. Results can be: no fermentation; acid only; or acid and gas.
Fermentation is a valuable tool in the identification of certain species of bacteria - variety of end products.
- Absence of external electron acceptors
- Carbon atoms of organic compounds are only partially oxidized
Carbohydrate Fermentation Test: results
This test indicates the ability of the microorganism to break down gelatin, a protein. If the bacteria has the enzyme gelatinase, the medium turns from a solid to a liquid. If the bacteria does not have the enzyme, the medium remains a solid. If the medium was incubated at 37C, it will need to be refrigerated before determining the outcome of the test.
Starch Agar test
Starch in a nutrient agar
This test indicates the microorganism's ability to break down starch via the exoenzyme amylase. Iodine is added to the agar plate following incubation of the culture. If amylase was secreted, there is nothing for the iodine to stain, and there is a clear space around the bacterial colonies. If amylase was not secreted, the iodine stains the starch dark purple to black around the bacterial colonies.
Casein Agar Test
Milk in a nutrient agar
This test indicates the microorganism's ability to break down casein via a protease exoenzyme. If the protease was secreted, there is a clear space around the bacterial colonies. If the protease was not secreted, there is no clearing around the colonies.
Spirit Blue Agar test
Tributyrin in a nutrient agar
This test indicates the microorganism's ability to break down triglycerides via a lipase exoenzyme. If the lipase was secreted, there is a dark blue precipitate around the bacterial colonies. If the lipase was not secreted, there is no dark blue pigmentation around the colonies.
DNA Agar test
a. DNA in a nutrient agar
This test indicates the microorganism's ability to break down DNA via a DNase. 1.0 M HCl is added to the agar plate following incubation of the culture. If DNase was secreted by the bacteria, there will be no DNA to react with the HCl, and there will be a clear space around the bacterial colonies. If DNase was not secreted, the DNA will still be present and will be precipitated out of solution in the agar by the HCl causing the agar to appear cloudy.
Citrate Agar test
Citrate is the only carbon containing compound in the medium
Utilization of citrate as the sole carbon source is indicated by a color change from green to blue. As the microorganism metabolizes the citrate, it releases ammonia as waste products, and causes the pH to become alkaline. The pH change causes the pH indicator, bromphenol blue to change from green to blue.
Urea in a nutrient broth or nutrient agar slant
This test indicates the microorganism's ability to break down urea into ammonia and CO2 by the enzyme urease. The urea broth with phenol red as the pH indicator turns hot pink (fuschia) in color due to the presence of the ammonia from the breakdown of the urea.
Catalase converts hydrogen peroxide to water and oxygen. Usually facultative anaerobes, obligate aerobes and microaerophiles have catalase.
Catalase test results
H2O2 is added directly to a pure culture on solid media or in broth
This test indicates the presence of the enzyme catalase in the microorganism. If the enzyme is present, bubbles (of O2 gas) are liberated after adding H2O2.This test can be used to differentiate between aerobic (also facultative anaerobic) and some anaerobic bacteria.
Sulfide Indole Motility Medium (SIM) - multiple test media (MTM)
- H2S production is indicated by a black precipitate
- Indole is indicated by a cherry-red color after adding Kovac's reagent.
- Motility is indicated by growth away from the stab line in the agar.
This test indicates the ability of the microorganism to metabolize the amino acid cysteine with the release of H2S which reacts with the ferric compound in the medium to produce a black precipitate. This test also indicates the microorganism's ability to break down the amino acid tryptophan, giving off indole as a waste product which is identified using Kovac's reagent. And, it also indicates the microorganism's motility by the pattern of growth.
Methyl Red Fermentation: ingredients
a. Glucose is the only sugar in the nutrient broth.
b. Mixed-acid production is indicated by the broth turning red after adding Methyl Red dye.
Methyl Red Results
This test indicates a mixed-acid fermentation of glucose by the bacteria. In a positive response, the amount of acid produced by the bacteria is enough to lower the pH of the media to <4.8. Five drops of methyl red are added to the culture medium. In a positive response the medium turns red, while in a negative response it does not.
VP - mixed acid test
Glucose is the only sugar in the nutrient broth.
2,3-butanediol production is indicated by the broth turning red after adding alpha-naphthol (Barritt's reagent A) and KOH (Barritt's reagent A) and shaking vigorously. This test indicates that glucose is fermented producing mainly a pH neutral end product - 2,3-butanediol. Barritt's reagents detect the presence of acetoin. Acetoin - a precursor of 2,3-butanediol. Positive reactive - red cloudiness at the top.
Phenylamine Agar Slant
The amino acid phenylalanine is incorporated into the medium.
Degradation of phenylalanine is indicated by a green color after adding 10% ferric chloride. If the bacteria has the enzyme phenylalanine deaminase, it breaks down phenylalanine into phenylpyruvic acid and ammonia. 10% ferric chloride is added to the medium which reacts with the phenylpyruvic acid to produce a green colored complex.
Tryptone broth: Ingredients
- The amino acid tryptophan (in high concentration) is incorporated into the nutrient broth.
- Indole is indicated by a cherry-red color after adding Kovac's reagent.
Indole is indicated by a cherry-red color after adding Kovac's reagent.
This test detects the degradation of the amino acid tryptophan by the enzyme tryptophanase, which results in the production of indole and pyruvic acid. The pyruvic acid is further metabolized and the indole is given off as a waste product. Ten drops of Kovac's reagent are added to the medium. In a positive response the Kovac's reagent changes color from yellow to red, while in a negative response there is no color change.
KIA test: Ingredients
The fermentation of both glucose and lactose can be determined.
H2S production is indicated by a black precipitate
This test detects the fermentation of glucose and lactose. KIA contains: a) two carbon sources - glucose and lactose in different concentrations; b) peptone - amino acids and partially digested polypeptides; c) ferrous salts; d) pH indicator phenol red
KIA Test results
Alkaline slant (red)/acid butt (yellow) indicates that only glucose was fermented. Acid slant/acid butt (both yellow) indicates that both sugars were fermented. Alkaline slant/alkaline butt (both red) indicates that neither sugar was fermented. If gas is produced, there will be breaks or bubbles in the agar. This test also indicates the ability of the microorganism to metabolize the amino acid cysteine with the release of H2S which reacts with the ferric compound in the medium to produce a black precipitate.
- Thioglycollate and l-cysteine act as reducing agents to remove O2 from the medium.
- Resazurin acts as an oxygen indicator turning pink in the presence of O2. A yellow color /no color indicates the absence of O2.
This test helps to indicate the oxygen requirements of an organism. An obligate aerobe will grow at the top, an obligate anaerobe will grow at the bottom, and a facultative anaerobe and an aerotolerant anaerobe will grow everywhere.
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