Bio 139 Lab Exam 2

What is an indicator species?  What characteristics do we use to define an indicator species?

indicator species → a species that provides information about the environmental conditions of a particular ecosystem.

4 characteristics of an indicator species:

1. Exists in large numbers in feces of humans and other mammals

2. Cannot replicate outside of the mammalian host

3. Survives in water for the same amount of time as known fecal pathogens

4. Detected by simple and inexpensive methods

What is the purpose of using Colilert and the E. coli / Coliform Petrifilm in our water testing experiment of water collected from the American River?

Colilert and Coliform petrifilm help in detection of coliform bacteria (like E. coli) in water samples from the American River.

• Purpose → to assess water quality and identify potential fecal contamination, which is crucial for human and environmental health.

For Petrifilm, what do the colors indicate? What are the benefits of using petrifilm?

Petrifilm —> culture medium used for detecting and enumerating E. coli and environmental coliforms in water samples.

• Blue colonies = presence of E. coli due to β-glucuronidase reacting with BCIG.

• Red colonies = presence of environmental coliforms due to β-galactosidase reacting with MUG.

• No color change or other colors indicate absence of target bacteria.

Benefits:

• ability to quantify colonies

For Colilert, what does UV fluorescence indicate? What are the benefits of using Colilert?

Colilert —> a defined substrate method used for detecting and enumerating total coliforms and E. coli in water samples.

• UV fluorescence = the presence of E. coli coliforms.

  • E. coli colonies fluoresce when exposed to UV light due to reaction of MUG and beta-glucuronidase.

• Yellow color = presence of any coliforms

  • colilert expresses yellow color due to reaction of ONPG and beta-galactosidase.

Benefits:

• Fast and accurate detection of environmental coliforms and E. coli coliforms

• bigger amount used (as opposed to petrifilm) so bigger chance of detecting any specific coliforms

In the Colilert, what enzymes are being detected to indicate the presence of the following groups and which reagents are responsible for the detection?

• Coliforms:

  • Enzyme Detected: β-galactosidase

  • Reagent: ONPG (ortho-Nitrophenyl-β-galactoside)

  • Result: Hydrolysis of ONPG by β-galactosidase produces a yellow color.

• E. coli coliforms:

  • Enzyme Detected: β-glucuronidase

  • Reagent: MUG (4-methylumbelliferyl-β-D-glucuronide)

  • Result: Hydrolysis of MUG by β-glucuronidase produces fluorescence under UV light.

What are the active ingredients in E.coli / coliform Petrifilm? How do they lead to identification of environmental and fecal coliforms?

• tetrazolium → tetrazolium is reduced to formazan by enzymes present in coliform bacteria, leading to colony formation.

• BCIG → BCIG reacts with enzyme β-glucuronidase (specific to E. coli) forming the blue color.

How does the KOH test allow you to distinguish between Gram positive and Gram negative cells?

• Gram-negative cells heaven outer membrane which allows them to release their DNA when KOH is added, causing the solution to lyse

• Gram-positive cells lack an outer membrane, so the solution remains liquid.

What are endospores and how are they detected using the endospore staining technique?

Endospores → dormant, resistant structures formed by certain bacteria.

• Detection: Heat-fixed bacterial smears are stained with malachite green, washed, and counterstained with safranin. Endospores appear green, while vegetative cells are pink.

• Can be terminal, subterminal, and centered endospores

Recall how the gram staining procedure works to and how it allows you to distinguish between Gram positive and Gram negative cells.

• Gram negative cells have a thinner layer of peptidoglycan, so when primary stained, decolorized, the primary stain is removed off of the gram negative cells, but not the gram positive. The gram negative will appear pink and the gram positive will remain purple which will help us distinguish the two

What is MacConkey agar? Why do we use it? What are its selective and differential components?

MacConkey Agar —> a selective and differential culture medium used in microbiology.

• Use: used as a selective and differential medium used to isolate and differentiate lactose-fermenting Gram-negative bacteria, particularly enteric pathogens.

• Differential Agents: Lactose, neutral red pH indicator.

  • Lactose fermentation leads to the production of acidic byproducts, causing a drop in pH.

  • Neutral red turns pink/red when the pH drops below a certain level, indicating lactose fermentation.

  • Non-fermenters appear colorless.

• Selective Agent: Crystal violet, bile salts.

  • inhibit the growth of Gram-positive bacteria and select for Gram-negative bacteria.

What is Nutrient agar? Why do we use it?

Nutrient agar —> a general-purpose growth medium used to cultivate a wide range of microorganisms.

• Use: Used to provide essential nutrients for microbial growth and uses Agar as a solidifying agent to solidify the medium for bacterial growth.

• Not selective or differential; simply supports the growth of a variety of microorganisms without inhibiting any specific group.

Define the following:

• obligate aerobes

• aerotolerant microbes

• facultative anaerobes

• obligate anaerobes

• Obligate Aerobes → Microorganisms that require oxygen for growth and can only survive in aerobic environments.

• Aerotolerant Microbes → Microorganisms that can tolerate the presence of oxygen but do not require it for growth.

• Facultative Anaerobes → Microorganisms that can grow in the presence or absence of oxygen; can switch between aerobic respiration and fermentation depending on oxygen availability.

• Obligate Anaerobes → Microorganisms that cannot tolerate the presence of oxygen and can only grow in the absence of oxygen.

What is the Catalase Test used for? What is the reaction? What does the bubbling indicate?

Catalase test —> test that helps detect the presence of the enzyme catalase in bacterial cells.

• Reaction: Catalase breaks down hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2).

• Bubbling after adding hydrogen peroxide indicates a positive catalase reaction, confirming the presence of catalase enzyme in the bacterial cells.

Procedure: 

1. A small amount of bacterial culture is mixed with hydrogen peroxide solution on a slide.

2. Observation for the rapid formation of bubbles indicates a positive catalase reaction. Absence of bubbling indicates a negative catalase reaction

What is the Oxidase Test used for? What is the reaction?

Oxidase test —> test that helps detect the presence of cytochrome c oxidase enzyme in bacterial cells.

• Cytochrome c oxidase is involved in the electron transport chain and catalyzes the transfer of electrons to oxygen during aerobic respiration.

• Reaction: Oxidase test utilizes a chromogenic reagent (like TMPD) which turns purple in the presence of cytochrome c oxidase.

Procedure:

1. A small amount of bacterial culture is applied to a filter paper or swab.

2. The filter paper or swab is then moistened with oxidase reagent containing TMPD.

3. Development of a purple color within 10-30 seconds indicates a positive oxidase reaction, confirming the presence of cytochrome c oxidase in the bacterial cells. Absence of color change within the specified time indicates a negative oxidase reaction.

For BCP Sugars Test, what are the 1) reactants 2) products 3) how products are detected, and 4) what negative or positive results look like and what they mean.

BCP sugars test —> a biochemical test used to detect the fermentation of sugars by bacteria.

1. Reactants:

   • Bacterial culture

   • Bromocresol Purple (BCP) indicator

   • Sugars —> glucose, lactose, and sucrose

2. Products:

   • Acidic byproducts (from sugar fermentation)

3. Detection of Products:

   • Bromocresol Purple (BCP) changes color in response to pH changes.

     • acidic environment —> yellow

     • basic environment —> purple.

4. Interpretation of Results:

   • Positive Result: turns yellow = acid production due to sugar fermentation, resulting in a decrease in pH.

   • Negative Result: remains purple = no fermentation of sugars and thus no acid production.

For Indole Test, what are the 1) reactants 2) products 3) how products are detected, and 4) what negative or positive results look like and what they mean.

Indole test —> a biochemical assay used to detect the ability of bacteria to produce indole from the amino acid tryptophan

1. Reactants:

   • Bacterial culture

   • Tryptone broth or other media containing tryptophan.

2. Products:

   • Indole produced from tryptophan metabolism

3. Detection of Products:

   • Kovac's reagent, which contains p-DMABA, is added to the bacterial culture.

   • A positive test is indicated by the formation of a red ring at the air-liquid interface.

4. Interpretation of Results:

   • Positive Result: Presence of a red ring = production of indole due to tryptophan metabolism.

   • Negative Result: Absence of a red ring = no indole production

For the Voges-Proskauer (VP) test, what are the 1) reactants 2) products 3) how products are detected, and 4) what negative or positive results look like and what they mean.

The Voges-Proskauer (VP) test —> a biochemical assay used to detect the production of acetoin by bacteria. Here's how it works:

1. Reactants:

   • Bacterial culture

   • MRVP broth, containing glucose as the carbohydrate source

2. Products:

   • Acetoin produced from glucose metabolism via the 2,3-butanediol fermentation pathway

3. Detection of Products:

   • Potassium hydroxide (KOH) is added to the bacterial culture, which oxidizes acetoin to diacetyl.

   • Diacetyl then reacts with guanidine nuclei from peptone in the medium, producing a dark red color.

4. Interpretation of Results:

   • Positive Result: Development of a dark red color = presence of acetoin, suggesting 2,3-butanediol fermentation.

   • Negative Result: Absence of a color change or change to a copper color = no acetoin production.

For the Methyl Red Test, what are the 1) reactants 2) products 3) how products are detected, and 4) what negative or positive results look like and what they mean.

The Methyl Red (MR) test —> a biochemical assay used to detect mixed-acid fermentation of glucose by bacteria.

1. Reactants:

   • Bacterial culture

   • MRVP broth, containing glucose as the carbohydrate source

2. Products:

   • Stable acidic end products (lactate, acetate, and formate) produced from glucose metabolism via mixed-acid fermentation

3. Detection of Products:

   • Methyl Red indicator is added to the bacterial culture.

   • If the pH of the medium drops to 4.4 or below due to the acidic products, the indicator turns bright red.

4. Interpretation of Results:

   • Positive Result: Development of a bright red color = the presence of stable acidic products, suggesting mixed-acid fermentation.

   • Negative Result: Absence of a color change or change to shades of orange or yellow = no or insufficient production of stable acidic products.

For the Nitrate Reduction/Denitrification Test, what are the 1) reactants 2) products 3) how products are detected, and 4) what negative or positive results look like and what they mean.

The Nitrate Reduction/Denitrification Test —> a biochemical assay used to detect the ability of bacteria to reduce nitrate to nitrite and further to other nitrogenous compounds.

1. Reactants:

   • Bacterial culture

   • Nitrate broth containing potassium nitrate as a source of nitrate

2. Detection of Nitrate Reduction:

   • After incubation, the culture is checked for gas production in the Durham tube.

   • Gas production indicates the reduction of nitrate past nitrite during anaerobic respiration, confirming the presence of denitrification.

   • Gas production by known fermenters leads to inconclusive results as it's unclear how the gas was produced.

3. Detection of Nitrite:

   • If no gas is observed or if gas is produced by known fermenters, sulfanilic acid and α-naphthylamine are added to the medium.

   • Nitrite reacts with these indicator substrates to form a red azo dye, indicating nitrate reduction to nitrite.

4. Confirmation with Zinc:

   • If a red dye is not produced, zinc powder is added to the medium.

   • Zinc converts any remaining nitrate to nitrite, which reacts with the indicators to produce a red azo dye.

   • Tubes that remain colorless after zinc addition indicate nitrate was reduced past nitrite to other nitrogenous compounds, confirming denitrification.

For the Citrate Agar test, what are the 1) reactants 2) products 3) how products are detected, and 4) what negative or positive results look like and what they mean.

The citrate agar test —> a biochemical assay used to determine the ability of bacteria to utilize citrate as the sole carbon source.

1. Reactants:

   • Bacterial culture

   • Citrate agar medium containing sodium citrate as the sole carbon source, ammonium phosphate, and bromthymol blue indicator.

2. Detection of Citrate Utilization:

   • If bacteria possess the enzyme citrate permease, they can transport citrate into the cell and metabolize it, leading to the alkalization of the medium.

   • Bromthymol blue dye in the medium changes from green (neutral pH) to blue (alkaline pH) when organisms utilize citrate.

3. Interpretation of Results:

   • Positive Result:

     • Growth of bacteria on citrate agar medium, indicated by a change in color of the medium from green to blue.

   • Negative Result:

     • No growth of bacteria or no change in the color of the medium, indicating the inability of bacteria to utilize citrate.

For the Motility agar test, what are the 1) reactants 2) products 3) how products are detected, and 4) what negative or positive results look like and what they mean.

The motility agar test —> a biochemical assay used to determine the motility of bacteria, which refers to their ability to move actively through a medium or on a surface.

1. Medium Composition:

   • Motility agar has a low concentration of agar which allows bacteria to move through the medium instead of than only the surface.

2. Inoculation:

   • Bacterial culture is inoculated into the center of a motility agar tube using a sterile inoculating needle or loop.

3. Observation:

   • After incubation, motile bacteria will move outward from the point of inoculation, creating “flare”-like growth patterns from needle stab.

   • Non-motile bacteria will only show growth along the stab-line without radiating flares.

4. Interpretation of Results:

   • Positive Result:

     • Presence of visible flaring growth patterns extending from needle stab

   • Negative Result:

     • Absence of radiating growth patterns, indicating no motility.

   • NOTE: bacteria that exhibit motility on motility agar are often equipped with flagella (or other motility structures) which allow them to move through the medium.