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Questions and Answers
Which of the following is NOT a characteristic of Enterobacteriaceae?
Which of the following is NOT a characteristic of Enterobacteriaceae?
Which of the following bacterial species is a primary pathogen?
Which of the following bacterial species is a primary pathogen?
Which of the following is NOT a selective and differential media?
Which of the following is NOT a selective and differential media?
Which of the following is a non-lactose fermenter?
Which of the following is a non-lactose fermenter?
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Which of the following media is designed to isolate and differentiate Salmonella and Shigella?
Which of the following media is designed to isolate and differentiate Salmonella and Shigella?
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Which of the following is NOT a common biochemical test used to identify Enterobacteriaceae?
Which of the following is NOT a common biochemical test used to identify Enterobacteriaceae?
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What does a black precipitate in Triple Sugar Iron (TSI) agar indicate?
What does a black precipitate in Triple Sugar Iron (TSI) agar indicate?
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What does a yellow butt and a yellow slant in Triple Sugar Iron (TSI) agar indicate?
What does a yellow butt and a yellow slant in Triple Sugar Iron (TSI) agar indicate?
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Which of the following tests detects the production of mixed acids from glucose because of subsequent metabolism of pyruvate?
Which of the following tests detects the production of mixed acids from glucose because of subsequent metabolism of pyruvate?
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A gram-negative bacillus that produces an acid slant and acid butt on triple sugar iron agar can ferment which of the following carbohydrates?
A gram-negative bacillus that produces an acid slant and acid butt on triple sugar iron agar can ferment which of the following carbohydrates?
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Tryptophan broth is inoculated and incubated 24 hours. After incubation, Kovac's reagent is added. A red color develops at the surface of the broth. Which product of metabolism was formed?
Tryptophan broth is inoculated and incubated 24 hours. After incubation, Kovac's reagent is added. A red color develops at the surface of the broth. Which product of metabolism was formed?
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Indole-positive bacteria:
Indole-positive bacteria:
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What type of ID method is employed by the API system?
What type of ID method is employed by the API system?
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What do black colonies on XLD agar indicate?
What do black colonies on XLD agar indicate?
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What do orange colonies on HEK agar indicate?
What do orange colonies on HEK agar indicate?
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Most pathogenic Enterobacteriaceae are:
Most pathogenic Enterobacteriaceae are:
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Pink (lactose fermenting) colonies on SS agar could be:
Pink (lactose fermenting) colonies on SS agar could be:
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Study Notes
Lecture 9 - Overview of Identification Methods for Enterobacteriaceae
- Enterobacteriaceae is a family of bacteria, often called enterics, containing many genera and species.
- Clinical isolates in acute-care settings are primarily Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis.
- Common identification methods include using special media, various tests, and test systems.
Introduction to Enterobacteriaceae
- Enterobacteriaceae encompasses a large group of bacteria.
- Clinical isolates often include species like Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis.
- These bacteria are commonly found in acute care settings.
Clinical Significance
- Opportunistic pathogens: Enterobacteriaceae can cause infections in sites other than normally their natural environment, like the gut. Examples include Escherichia coli infections in wounds, urinary tract infections (UTIs), or meningitis.
- Primary pathogens: Some Enterobacteriaceae species are always pathogens. Examples include Salmonella spp., Shigella spp., and Yersinia spp., which cause specific illnesses like diarrhea or plague.
General Characteristics of Enterobacteriaceae
- All ferment glucose.
- All reduce nitrates to nitrites.
- All are oxidase-negative.
- Most are motile at body temperature, except Klebsiella, Shigella, and Yersinia.
Enterobacteriaceae - ID
- Cellular morphology is not very useful (gram-negative rods).
- Growth conditions are often facultatively anaerobic.
- Modern methods for identification include molecular methods and different cultural techniques.
- Macroscopic morphology on BAP (Blood Agar Plate) may not be useful beyond ruling out other organisms for further identification.
- Often use selective plates to identify pathogenic species, such as those found in stool specimens.
MacConkey Agar
- Enterobacteriaceae can grow on MacConkey agar.
- MacConkey agar is selective and differential.
- Bile salts and crystal violet inhibit gram-positive organisms.
- Lactose fermentation differentiates colonies into pink (lactose fermenters) or yellow (non-lactose fermenters). Late lactose fermenters might appear as yellow at 24 hours but become pink at 48 hours.
- Lactose fermenters are a particular type.
EMB Agar
- Eosin-methylene blue agar (EMB) is a selective and differential medium.
- Methylene blue inhibits gram-positive bacteria.
- Lactose and sucrose fermentation are used in differential identification.
- EMB is particularly useful for lactose and sucrose fermenters.
Hektoen Enteric Agar (HE)
- HE agar is selective and differential medium, specifically designed for Salmonella and Shigella.
- Bile salts and other ingredients inhibit some gram-positive and gram-negative bacteria.
- Lactose and sucrose are ingredients for testing differential growth.
- Colonies appear either orange with low pH or blue/green for the target pathogens, with a black center for Salmonella.
Xylose Lysine Deoxycholate Agar (XLD Agar)
- XLD agar is designed for differentiating Salmonella and Shigella from other bacteria.
- It is selective and differential.
- Sodium deoxycholate inhibits the growth of gram-positive bacteria.
- Xylose, lysine, and sucrose and lactose are for differential growth.
- Phenol red is an indicator of pH changes.
- Black colonies indicate target pathogens, e.g., Salmonella. Yellow colonies often indicate nonpathogens.
Salmonella-Shigella Agar (SS Agar)
- Selective for Salmonella and Shigella.
- Bile salts, sodium citrate, and brilliant green inhibit other species, mainly gram-positive organisms and some lactose fermenting gram-negative rods
- Lactose fermentation is one of the differentials to separate organisms.
- Usually used to distinguish pathogenic species (e.g., Salmonella and Shigella) from common stool flora.
CIN Media
- Cefsulodin, irgasan, novobiocin (CIN) media is designed for isolating Yersinia spp.
- Inhibitor agents, like cefsulodin, irgasan, novobiocin, bile salts, and crystal violet, inhibit other bacterial species.
- Mannitol with phenol red are differential ingredients.
Selenite Broth
- Selectively inhibits gram-positive and many gram-negative bacteria.
- Enriches the growth of Salmonella spp.
- Some Shigella can also grow in Selenite broth.
- Primarily used for suspected pathogens occurring in low concentrations in stool samples and to enrich the growth of these pathogens.
Other Media for Salmonella
- Brilliant Green Agar is selective for Salmonella.
- It utilizes Brilliant Green dye for inhibition of competing organisms and Salmonella colonies often form a white to pink or red halo.
- Bismuth Sulfite Agar is a selective and differential medium. This medium is particularly useful to identify Salmonella because the colonies appear black.
Biochemical Tests for Enterobacteriaceae
- A series of biochemical tests are used to further characterize isolates of Enterobacteriaceae.
- Common tests include oxidase, TSI (triple sugar iron), ONPG, decarboxylase (e.g., lysine, ornithine), citrate, hydrogen sulfide (H2S), and numerous others.
- These tests are used to distinguish between various Enterobacteriaceae species.
Lactose Utilization
- Lactose is a disaccharide requiring an enzyme that breaks it down into simple sugars.
ONPG Test
- ONPG test is used to determine if an organism ferments lactose and has the ability to use lactose as a fermenting substrate.
- The ONPG test uses a substrate similar to lactose (called Ortho-Nitrophenyl-β-D-galactopyranoside) and tests for the presence of the enzyme beta-galactosidase.
Glucose Fermentation
- This process involves certain biochemical pathways (Embden-Meyerhof-Parnas Pathway and the Entner-Doudoroff Pathway) that generate different by-products.
- The byproduct analysis can be used to identify particular bacterial species.
Oxidation-Fermentation (O/F) Tests
- Used to identify oxidation or fermentation patterns of certain bacteria based on sugar utilization in aerobic and anaerobic conditions.
- O/F tests use an O/F basal media to check for the presence of various by-products from carbohydrate metabolism.
O/F Basal Media (OFBM)
- OFBM is used to identify whether certain species of bacteria are oxidizers or fermenters.
- Testing is done by inoculating two tubes, one of which has been overlaid with sterile oil.
- Acid is often identified as a color change.
Triple Sugar Iron Agar (TSI)
- A culture media specifically designed to detect and identify glucose, lactose, and sucrose fermentation, and hydrogen sulfide production.
- This medium is used initially to determine what type of sugars and metabolic pathways the bacterium under test might have.
Methyl Red (MR) Test
- Detects the production of mixed organic acids during glucose fermentation.
- MR testing can determine whether a certain species of bacteria uses a mixed acid fermentation pathway.
Voges-Proskauer (VP) Test
- Detect specific by-products of certain fermentation pathways.
- VP test used to determine if particular species of bacteria use a butylene glycol fermentation pathway.
Decarboxylase and Dihydrolase Tests
- These tests focus on identifying specific enzymes that remove a carboxyl group from amino acids by species of bacteria.
Phenylalanine Deaminase Test
- Determines the ability of a certain species of bacteria to deaminate a specific amino acid derivative, phenylalanine, into another compound.
Tryptophan Deaminase Test
- This test specifically assesses the ability of bacteria to deaminate tryptophan, often used in identification of certain bacterial species.
Citrate Utilization
- Measures an organism's capacity to use sodium citrate as a sole carbon source.
- The test evaluates whether a particular type of bacteria utilizes sodium citrate as a primary carbon source.
Indole Test
- This test is designed specifically to identify whether certain species of bacteria can produce indole.
Motility-Indole-Ornithine (MIO) Agar
- Semi-solid medium utilized to determine motility as well as detect and identify ornithine decarboxylase and indole.
- Used for differentiating between species such as Klebsiella, Enterobacter, and Serratia.
Nitrate and Nitrite Reduction
- Nitrate and nitrite reduction tests look at the ability of a certain species of bacteria to reduce nitrates into nitrites.
- If the species of bacteria can reduce nitrates to nitrites, no color change is observed, and no gas is produced. If it cannot reduce nitrates to nitrites, a color change to a red color may be observed and gas may be produced.
Urease Test
- Measures the capacity of an organism to produce urea, an enzyme significant for certain species of bacteria.
- The test involves identifying the ability of an unknown species of bacteria to use an enzyme called urease to hydrolyze urea.
Multi-test Systems
- Include various methods for identification of microorganisms, which includes different types of systems that may be automated or manual.
Rapid Identification Systems
- A faster alternative to traditional microbiological methods for identifying bacteria, usually a few hours versus many days.
- Different systems exist and employ varying methods, such as enzyme immunoassays and others, often aimed at giving rapid and reliable results.
Evaluation of Identification Systems
- Evaluating automated methods against standard "gold standard" methods to compare accuracy, cost-effectiveness, and workflow.
Knowledge Checks (Questions and Answers)
- Various questions about specific tests, and microbial characteristics used for the purpose of testing.
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