Micro Lab Exam #2
Oxygen Requirements
Obligate Aerobe: Only grows at atmospheric levels of oxygen. Obligate Anaerobe: Only grows in the ABSENCE of oxygen. Facultative Anaerobe: Can grow with or without oxygen, but grows better in the presence of oxygen. Microaerophile: Requires oxygen, but less than 20%. Aerotolerant: Grows equally well with or without oxygen.
Lysine Iron Agar
to compare to 2 make sure there is black on top
BCP sugars test
-Recall that Bromo Cresol Purple, the color indicator, will turn yellow if the pH is lowered from 6.8 to ~5.2 (cause: acidic fermentation products) -The durham tube will capture insoluble H2 gas due to fermentation
Oxygen Requirement
ATP generated by the pathway intermediates and products of cellular respiration(oxidative and substrate-level phosphorylation) and fermentation (substrate-level phosphorylation only) sustain microbial existence. Use of molecular oxygen (O2) as the terminal electron acceptor (TEA) for the electron transport chain during aerobic cellular respiration provides the highest metabolic energy yield, but it is often imperative that ATP is generated by the anoxic processes of anaerobic cellular respiration and fermentation because: (1) bacteria are not equally equipped with mechanisms to utilize O2 and; (2) environmental levels of O2 vary or are absent altogether. Interestingly, redox balance in anaerobic respiration is provided by alternative TEAs including NO3- and SO4-, while the pathway intermediate pyruvate serves the same function during anoxic fermentation. Bacteria can be categorized into one of five groups based on O2 requirements. Obligate aerobes respire only using oxygen as a TEA. Microaerophiles respire only using oxygen as a TEA but grow best at reduced oxygen levels. Facultative anaerobes are capable of aerobic cellular respiration as well as fermentation. Aerotolerant organisms ferment only. Obligate anaerobes die in the presence of oxygen, so they must use alternative electron acceptors for cellular respiration or perform fermentation for ATP production. Native bacteria living in the American River have adapted to different oxygen environments. High levels of oxygen (atmospheric oxygen, 20.8%) are associated with surface waters, while lower than atmospheric levels of oxygen exist in river sediment. In deep sediments oxygen levels approach 0.0%. Coliforms living in the human gut have adapted to differing oxygen levels that exist in the gastro-intestinal tract, performing cellular respiration using oxygen as a TEA when oxygen is available, or anaerobic metabolism (respiration or fermentation) when oxygen is limited. Coliforms are defined as Gram negative facultatively anaerobic non-spore forming rods that produce acid and gas while fermenting lactose during incubation at 37º C for 48 hours.
Candle Jar/Anaerobe jar
Anaerobe Jar has 0-0.5% O2, Candle jar 6-8% O2 , Atmosphere 21% O2 .
Nitrate Reduction and Denitrification
Bacteria can be tested for the presence or absence of nitrate reducing enzymes and their associated products following growth in a broth containing peptones as a nutrient source and potassium nitrate as a TEA. Following Incubation for Growth: Step 1: Inspect growth in nitrate broth for the presence of denitrification. A bubble in the Durham tube produced by a known non-fermenter confirms that nitrate was reduced past nitrite during anaerobic respiration and the test is complete. Gas produced by known fermenters represents an inconclusive result because there is no way to distinguish how this product was made. Step 2: If no N2 gas is observed or gas is produced by a known fermenter in step 1, indicator substrates sulfanilic acid and α-naphthylamine are added to the media. These compounds will react with the first metabolic product of dissimilative nitrate reduction, nitrite, to form a red azo dye. This is a positive test for nitrate reduction (NO3 - to NO2 - ). A lack of color change means that either: (1) nitrate was not reduced to nitrite or; (2) nitrate was reduced to nitrite, and nitrite was reduced further to other denitrification products in the chart above that do not react with the indicator substrates. Step 3: If a red dye is not produced in step 2, zinc powder is added to the media. Zinc converts nitrate to nitrite, which will react with the indicator substrates sulfanilic acid and α-naphthylamine that were added during step 2 to produce a red azo dye. The presence of nitrate that triggers this reaction indicates that the bacteria did not reduce it while growing in the media and is therefore a negative test for nitrate reduction. Tubes that remain colorless after zinc addition indicate that nitrate was reduced to NO, N2O, or some other nongaseous nitrogenous compound and therefore represent a positive denitrification test.
Endospore Isolation and Staining
Bacteria function as open systems and are thus capable of sensing and responding to environmental changes. One example of a survival strategy used by certain soil-dwelling Gram positive bacteria such as Bacillus spp. is the formation of endospores during a process called sporulation. This complex developmental cycle occurs when metabolically active vegetative cells are triggered by a stressor such as nutrient deprivation to produce a dormant, non-reproductive endospore that preserves cellular genetic material and vital proteins. Endospores are resistant to extreme conditions that would normally kill the vegetative cell including exposure to high temperature, UV irradiation, desiccation, and chemicals such as detergents, antibiotics, and acids. Upon death of the vegetative cell, endospores are released into the environment and easily dispersed by wind and water, where they can remain stable for thousands of years. The return of favorable conditions triggers endospores to germinate back to vegetative cells in as little as 90 minutes.
The Endospore Stain: A type of Differential Stain
Differential Stain Primary Stain=malachite green Needs steam to permeate the endospore coats. Decolorizer=water Washes away Malachite green from vegetative cells. Only the endospores will remain green after this step Counter Stain=Safranin Vegetative cells will stain pink.
environmental vs fecal coliforms
Environmental coliforms: Occupy diverse niches including water, sediment, and vegetation; examples include Citrobacter, Enterobacter, and Klebsiella Fecal coliforms: Restricted for growth within the mammalian gastrointestinal tract; the sole example is nonpathogenic Escherichia coli, which is shed to the environment in feces at concentrations reaching 1x108 bacterial cells per gram and survives in water for approximately 72 hours without replicating.
. Glucose (and other Sugars) Fermentation Test using Bromocresol Purple
Fermentation is an anaerobic form of carbohydrate catabolism in which an organic substrate is both an electron donor and electron acceptor, and ATP is generated exclusively by substrate-level phosphorylation. Bacteria species are highly diverse and as a result: (1) do not ferment all possible donor compounds and; (2) produce different products depending on the presence or absence of particular enzymeencoding genes. Common but variable end products include a variety of acids, alcohols, and/or gases such as CO2 (soluble) or H2 (insoluble). These metabolic differences can be detected following inoculation and subsequent incubation in liquid broth containing a fermentable sugar and digestible protein peptones. Acidic products are detected by the inclusion of a pH indicator such as bromocresol purple, which changes from purple at pH 6.8 to yellow when the media is acidified below pH 5.2. Durham tubes are typically placed into culture vessels to collect any insoluble gases (e.g. H2) that may be produced. It is important not to let tubes incubate at 37ºC past 18 hours to avoid false negatives caused by a shift to alkaline pH (purple color) as a result of peptone deamination to ammonia (NH3) following exhaustion of the fermentable carbohydrate nutrient. This approach can also be used to detect neutral to alkaline end products (purple color) generated throughout the media by strictly respiring organisms that use an organic substrate as the electron donor and oxygen as the TEA. Interestingly, some classes of strictly respiring organisms generate acidic products (yellow color) observable at the air liquid interface.
IMViC tests
Indole, Methyl Red, Voges-Proskauer, Citrate identify genera within Enterobacteriaceae (which are: g-negative, facultative anaerobe, non spore forming rods)
cytochrome c oxidase
One such diagnostic determines whether bacteria metabolize using the ETC carrier enzyme cytochrome C oxidase. This test utilizes NNDP, an artificial electron donor, as the substrate which will be oxidized to a pink/purple product called indophenol by cellular respiration. The artificial electron donor is not oxidized and therefore remains colorless when added to bacteria lacking cytochrome C oxidase. Important examples include: Cytochrome C Oxidase Negative: Gram (-), rod-shaped enteric bacteria that grow harmlessly in the mammalian gastrointestinal tract. Species in the family Enterobacteriaceae are facultative anaerobes and key genera include Escherichia, Salmonella, etc Cytochrome C Oxidase Positive: Strictly respiring pathogenic genera of the families Pseudomonadaceae and Neisseriaceae. Fish pathogens belonging to the family Aeromonadaceae are facultative anaerobes. Organisms that test positive for cytochrome C oxidase use oxygen as a TEA during aerobic cellular respiration but are not necessarily obligate aerobes. Similarly, organisms that test negative for cytochrome C oxidase are not necessarily obligate anaerobes and can use oxygen as a TEA during cellular respiration by passing electrons through other Redox carrying enzymes in the ETC.
Colilert System
The United States and 30 other developed nations worldwide test water samples for microbial growth by incubating them for 24 hours at 37º C in solution with Colilert, a proprietary mix containing: (1) inorganic nitrogen, sulfur, and phosphorus; (2) indicator substrates ortho-Nitrophenol-β-galactoside (ONPG) and 4-Methylumbelliferyl-β-D-glucuronide (MUG) which are the primary carbon sources and; (3) non-coliform inhibitors. All coliforms, including E. coli, produce the enzyme β-galactosidase to hydrolyze lactose for metabolism. ONPG structurally mimics lactose and this colorless compound is hydrolyzed to β-Dgalactopyranoside and o-Nitrophenol (ONP), the latter of which is bright yellow. However, E. coli, but not other coliforms, produces the enzyme β-glucuronidase to hydrolyze complex carbohydrates and this enzyme happens to hydrolyze the chemical MUG to β-D-glucuronide and 4-Methylumbelliferone (MU), the latter of which displays blue fluorescence under ultraviolet light. The water from positive tests using 100 mL water samples are further tested to estimate the most probable number (MPN) for total and fecal coliforms.
KOH Test
The significant difference in cell wall peptidoglycan content between Gram positive and Gram negative organisms can be detected by the potassium hydroxide (KOH) test. The KOH test is a secondary test to the Gram stain to determine peptidoglycan content. An alkali solution of 3% KOH will dissolve lipid bilayers including the cell (plasma) membrane of both Gram negative and Gram positive cells and the outer membrane of the Gram negative cell wall. In the presence of KOH on a glass slide, the membranes of Gram negative cells are disrupted. The hypotonic environment leads to a rise in osmotic pressure on, and subsequent rupture of, the thin peptidoglycan layer that remains. The lysed cells release intracellular contents onto the slide, and this can be observed by the attachment of sticky, viscous DNA to a metal inoculation loop. On the other hand, thick sheets of peptidoglycan in the Gram positive cell wall prevent lysis by a 3% KOH solution even though the cell membrane is dissolved since the KOH solution does not disrupt the peptidoglycan structure directly. Therefore, the KOH solution of a Gram positive sample will not visibly alter viscosity
The oxidase test
The test determines if cytochrome C oxidase is present in the electron transport chain of bacteria that can respire aerobically
Catalase Test using Hydrogen Peroxide
To counter the toxic H2O2, most obligate aerobic and facultative anaerobic bacteria produce catalase which catalyzes the conversion of 2 H2O2 → 2H2O + O2 It is therefore true that if a solution of commercial-grade 3% H2O2 is applied to catalase-positive organisms on a glass slide, bubbles will appear from the conversion of H2O2 to water and gaseous oxygen. Bacteria that respire aerobically generally utilize catalase to detoxify hydrogen peroxide as this process generates oxygen which can be utilized as the terminal electron acceptor for aerobic respiration, while organisms that do not respire aerobically utilize peroxidase to detoxify hydrogen peroxide which does not generate oxygen. The catalase test is primarily used in the clinical laboratory to distinguish among Gram positive cocci, but is also used to help define the metabolic classification of bacteria. The catalase test differentiates, in part, the Gram positive genera Micrococcus and Staphylococcus (catalase-positive) from Streptococcus (catalasenegative). For our purposes we will be utilizing the catalase test to help define the metabolic classification of your Gram negative bacteria.
Petrifilm System
Water samples can also be tested for microbial growth by incubating them for 24 hours at 37º C on a Petrifilm that contains indicator substrates tetrazolium and 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (BCIG). All coliforms, including E. coli, reduce tetrazolium during fermentation, which lowers the pH, produces gas, and turns colonies red in color. However, E. coli, but not other coliforms, produce the enzyme β-glucuronidase which will hydrolyze BCIG to β-D-glucuronide and 5-bromo-4-chloro-3-indole (BCI), the latter of which forms an insoluble blue precipitate in colonies. Positive tests from a 1 mL water sample are used to estimate CFU/mL for total and fecal coliforms. Keep in mind the level of detection using the Petrifilm system is lower compared to the Colilert test due to the smaller sample volume, but the Petrifilm advantage is the ability to use this test for density determination.
KOH test
if the organism is gram- the peptidoglycan layer is thin and pressure causes lyses KOH reacts with the DNA released from gram- cells to create a viscous mixture
