Practicum Review 2 PDF

Review all assigned readings in syllabus from lab manual and supplemental materials oQuizzes oPowerpoints onotes from lab (how to interpret tubes / plates) Study images of organisms (algae / protozoa / fungi & molds / bacterial staining & morphology) Make tables for all media tubes / plates, names o...

Review all assigned readings in syllabus from lab manual and supplemental materials oQuizzes oPowerpoints onotes from lab (how to interpret tubes / plates) Study images of organisms (algae / protozoa / fungi & molds / bacterial staining & morphology) Make tables for all media tubes / plates, names of classes of bacteria (e.g. mesophiles / thermophiles, enterics, coli types) Make tables for all metabolic tests including: oSubstrate oEnzyme oProduct(s) opH indicator / reagent added – name of assayed product if the primary product is converted oHow to interpret colorimetric results (indicators / presence of products etc.) – positive vs. negative for each test Note: This review is meant to supplement your studying, not stand alone. It may not include everything you are expected to know Brightfield Light gets blocked or absorbed/emitted by sample Direct light that doesn’t pass thru sample comes to eyes (bright) Staining needed for contrast ‐ organisms usually stained (not living) Darkfield Direct Light gets blocked by opaque disk (no direct light – dark) Only light refracted back to objective lens by specimen is visible Can observe unstained (living) organisms More contrast Phase‐Contrast No staining or fixation needed view live cells & structural details (within limit of resolution of visible light) uses phase differences for contrast: differences in refractive properties between the object and the aqueous environment è variations in brightness Uses 2 sets of phase rings to send phase‐shifted light to sample, sample changes phase, second set of rings used to convert the phase difference into difference in brightness (white / black / grey) Electron Microscope Uses electron beam to illuminate sample Very good resolution: Electrons have small wavelength (small λ – 0.0037nm) and extremely high N.A. equivalent as it is performed in a vacuum Resolving power is about 0.05nm vs. light about 200nm. All items to be viewed must be fixed with powerful fixatives, and labeled with gold particles. These gold particles deflect the electrons and create contrast on a phosphor plate (TEM) or deflection detectors and computer to assemble the image (SEM) Transmission electron microscope (TEM) used to visualize ultra‐thin sections (TEM of Bacillus subtilis) Scanning electron microscope (SEM) used to visualize complex surfaces (SEM of pollen) Lab 2 Wet mount, hanging drop techniques True motility vs. Brownian movement (not random, directed, overall positions are changing, ‐taxic) Algae and Protozoa Know the slide pictures on blackboard, prepared slides in class for weeks For every slide KNOW: Organism (species) name / phylum / Kingdom / Domain Special structures (e.g. heterocysts, cytostome, macronucleus) Mode of nutrition Mode of motility (if any) Colonial or non‐colonial If filamentous: branched or unbranched Conjugation, if occuring, recognize this Microorganisms With the exception of spirogyra, names are not required. I left the names on the slide because I find it difficult to include organisms without some identifying marks (I put strikethroughs on the names to emphasize that you will not be asked to name them - except maybe for spirogyra) Be able to identify type, characteristics and locomotion - e.g. protozoa, cilia; green algae, filamentous, colonial, unbranched Cyanobacteria (“blue green algae” NOT green algae) prokaryotic / Domain Bacteria, but they are often observed in water samples with algae identified according to their colors, storage products, and the chemical composition of their cell walls and flagella. Photosynthetic, (most) produce O2. Cyanobacteria: Anabaena (with heterocysts) Algae Eukaryotic Unicellular, filamentous or multicellular. Algae / Green algae: Cladophora Branched filaments, multinucleated cells Algae / Green algae: Pandorina (colonial) Colonial, flagellated Algae / Green algae: Spirogyra (vegetative) Spiral chloroplasts Filamentous unbranched Protozoa Protozoa Rhizopoda amoeba Ciliophora (ciliates) paramecium Euglenozoa Euglena Single celled eukaryotes/ No cell walls Motility: Amoeba ·Pseudopodia Euglena ·Flagellum Paramecium · Cilia Protozoa / Rhizopoda: Amoeba proteus (vital stain) Mode of locomotion pseudopodia Protozoa / Euglenozoa: Euglena Nucleus, eyespot/stigma, chloroplasts, flagellum visible Protozoa / Ciliophora: Paramecium Micro and macronucleus cilia lined oral groove Bacteria and Fungi Fungi: yeasts and molds Unicellular or multicellular eukaryotes Aerobic (except yeast) Sexual and Asexual spores (NOT to be confused with bacterial endospores which are not reproductive) Fungi: Molds/Yeast Budding Yeast - Asexual Conidia (spore holding structures) Conidiospores (ASEXUAL spores) Sporangia (spore holding structures) Sporangiospores (ASEXUAL spores) (dark dots) a. b. 3. C 4. D Fungi: Molds / Yeast Ascus (“sac” – spore holding structures) Ascospores (SEXUAL spores) Basidia (spore holding structures) Basidiospores (SEXUAL spores) Bacterial morphology & arrangement Bacillus (rod-shaped) Coccus (spherical-shaped) Spiral – Vibrio – Spirillum – Spirochete Star-shaped Rectangular Average size: 0.2 to 2.0 μm diameter × 2 to 8 μm length Most bacteria are monomorphic (single shape) A few are pleomorphic (many shapes) Pairs: diplococci, diplobacilli Clusters: staphylococci Chains: streptococci, streptobacilli Groups of four: tetrads Cubelike groups of eight: sarcinae Bacillus/Bacilli oBacillus has two meaning Bacillus Anthracis refers to a specific genus (anthrax pathogen). Bacillus means rod shaped; morphology of a cell E.coli can be described as bacillus, but it is not in the genus Bacillus, but with Eschericia genus Spirochete:(helical, 3 dimensional twist) Coccus/Cocci oMRSA oStreptococcus(chain of cocci) oStaphylococcus (cluster of cocci) Vibrio: 1 twist/turn Spirillum: Spiral, undulating in a plane Stains & Uses Simple Staining Simple stain: 1 reagent used, all bacteria appear similarly stained. Bacterial cell walls have slight negative surface charge, therefore: oDirect staining uses basic dyes (positive chromophore) oIndirect uses acidic dyes (negative chromophore) Direct stains with BASIC dyes Negative (Indirect) staining with acidic dyes Acidic stain: negatively charged chromophore (Nigrosin) o Will stain the background only, because the cell wall is also negatively charged (like charges repel) NO FIXATION IS USED (heat or methanol) Gives better idea of shape and size of cells than direct staining. Gram Stain Gram staining categorizes bacteria based on the physical and chemical structure of their outer surfaces: Gram‐positive bacteria have a thick layer made up of polymers of protein‐ sugar molecules (peptidoglycan). Gram staining of the peptidoglycan layer in the cell wall with crystal violet results in purple coloration of the gram‐ positive bacteria. Addition of acetone / alcohol dehydrates the peptidoglycan, causing it to retain the purple color. The CV decolorization rate is also slowed by the mordant iodine, which further helps it from escaping the cells in gram positives during decolorization. Gram‐negative bacteria have very thin peptidoglycan, and an outer membrane that is solubilized in the acetone/alcohol‐dehydration so instead loses the crystal violet stain completely during decolorization. At this point gram negative cells would be clear. A counterstain, made up of safranin, stains these bacteria reddish‐pink Gram‐Negative Coccus, clusters Gram‐Positive Coccus Spirillum (gram ‐) Gram‐Positive diplo‐bacillus Gram‐Negative Bacillus DIFFERENTIAL STAINS: ACID FAST Stain Some Gram positive cells also have MYCOLIC ACID in addition to the peptidoglycan layers. This is a waxy lipid. ACID FAST cells will retain a basic dye (carbolfuchsin) in the presence of acid‐ alcohol decolorization and are called acid‐fast. The carbolfuchin is more soluble in the waxy lipid mycolic acid than the acid‐alcohol used during decolorization Non–acid‐fast cells lose the basic stain when rinsed with acid‐alcohol, and are usually counterstained e.g. using methylene blue Families of ACID FAST bacteria: Mycobacteriaceae, Nocardiaceae, Gordoniaceae, Dietzaceae, Tsukamurellaceae. This technique is used to diagnose tuberculosis and leprosy (both caused by Mycobacteria) Lab 6: ELISA See the weekly posts Aseptic Technique EX. 10: Aseptic Transfer Protocol Microbiology Definitions Culture Media: a nutrient material prepared for the growth of microorganisms in a laboratory Inoculated / Inoculum: Microbes are introduced into a culture medium to keep them alive and study their growth. Aseptic Technique: Technique used to exclude contaminants Contamination: Presence of unwanted microbes Sterile: Containing no living microorganism Inoculating Loop & Inoculating Needle Pure Culture: Containing a single kind of microbe Bacterial Growth Requirements Chemical Requirements Nutrients – Culture Media Oxygen (O ) 2 Physical Requirements Temperature pH Osmotic Pressure Chemically Defined vs. Complex Media Chemically Defined Media: Exact chemical composition is known. Complex Media: Extracts and digests of yeasts, meat, or plants Nutrient broth Nutrient agar Agar Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C Different Types of Culture Media Broth - Provide large numbers of bacteria in a small space and easy to transport Ex) Nutrient broth Agar Slant - Containing solid culture media that were left at an angle while the agar solidified. Large surface area for growth is available. Ex) Nutrient agar slant Agar Deep - Agar solidifies in a vertical tube. Used to grow bacteria that prefer less O2. Ex) Semisolid Agar Deep: Containing 0.5%-0.7% agar, used to determine whether a bacterium is motile. Agar Plate - Containing 1.5% agar, solid culture media, provide large surface to isolate different microbes Ex) Nutrient agar plate Isolation of Bacteria & Special Media for Isolating Bacteria Different Dilution Techniques Streak Plate - Qualitative Technique Spread Plate Technique - Qualitative & Quantitative Technique Pour Plate Technique - Qualitative & Quantitative Technique Streak Plate Protocol 2. 1. Flame Take a loopful of culture Flame 4. 3. Flame Special Media for Isolation Enrichment Media - Contains chemicals that enhance the growth of desired bacteria Ex) Nutrient agar, Tryptic soy agar, Chocolate agar Differential Media - Differentiate colonies of desired microbes from others Ex) EMB(Eosin Methylene Blue), Blood,MacConkey Sabouraud agar Selective Media - Suppresses of unwanted microbes; encouraging desired microbes. Ex) PEA (PhenylEthyl Alcohol) Selective Media Phenylethyl alcohol agar (PEA) is a selective medium used to cultivate Gram positive organisms. The active ingredient, phenylethyl alcohol, inhibits or markedly reduces growth of Gram negative organisms by interfering with DNA synthesis. PEA also prevents Proteus species from swarming across the surface of the agar. Figure 6.9a Differential Media I EMB (Eosin Methylene Blue) 1. Coli-type colonies are very dark, almost black, when observed directly against the light. By reflected light a green sheen can be seen which is due to the precipitation of methylene blue in the medium from the very high amount of acid produced from fermentation. Those which form this type of colony are methyl red-positive lactose-fermenters such as most strains of E. coli and some strains of Citrobacter. Differential Media II EMB (Eosin Methylene Blue) 2. Aerogenes-type colonies are less dark. Often a dark center is seen surrounded by a wide, light-colored, mucoid rim – resulting in a "fish-eye" type of colony. Those which form this type of colony are methyl red-negative lactose-fermenters which include most strains of Klebsiella and Enterobacter. Differential Media III EMB (Eosin Methylene Blue) 3. Non-lactose-fermenting colonies produce no acid from fermentation, so the lighter-colored alkaline reaction is seen. Colonies of Pseudomonas (a strictly-aerobic non-fermenter) are shown below. Microbial Growth Microbial growth = increase in number of cells, not cell size Objectives 1. 2. 3. 4. 5. 6. 7. Differentiate between physical and chemical requirements for microbial growth Describe what happens in the population at each stage of the growth curve Differentiate between groups of bacteria classified according to their temperature tolerance Differentiate between groups of bacteria classified according to their oxygen tolerance Differentiate between groups of bacteria classified according to their pH requirements Describe how living cells react to solutions of varying tonicity. Describe two ways used in lab 8, of creating anaerobic conditions for culturing bacteria Tim and e betw e car multip en in ry o o l are ut m icatio culatio n not n div etabol - cells idin ism g. but Ra Po pid c ea pula ell g c t an h ge ion row d t d An un nera oub h. i int tibio form tion. les w for erfer tics ly st Cel ith ma e w eff ain ls s tio ith ect ed ma ll n. Gce ive. rollw rat wtha nu e inc lal lm En trien reas ost s e t typ dosp s, sp s - l tops. irr es p ores ace, ack o Dea eg ro fo et f th ula du rm c. c rs e ;s tai en om nin do e g tox ins ; Ce ac ll de c of umu aths nu la du tri tio e t en n o o ts. f tox ins , la ck Figure 6.14 Chemical Requirements - Oxygen Obligate aerobes are organisms that grow only in the presence of oxygen. They obtain their energy through aerobic respiration. Microaerophiles are organisms that require a low concentration of oxygen (2% to 10%) for growth, but higher concentrations are inhibitory. They obtain their energy through aerobic respiration. Obligate anaerobes are organisms that grow only in the absence of oxygen and, in fact, are often inhibited or killed by its presence. They obtain their energy through anaerobic respiration or fermentation. Aerotolerant anaerobes, like obligate anaerobes, cannot use oxygen to transform energy but can grow in its presence. They obtain energy only by fermentation and are known as obligate fermenters. Facultative anaerobes are organisms that grow with or without oxygen, but generally better with oxygen. They obtain their energy through aerobic respiration if oxygen is present, but use fermentation or anaerobic respiration if it is absent. Most bacteria are facultative anaerobes. Chemical Requirements - Oxygen Oxygen (O2) obligate aerobes Facultative Obligate anaerobes anaerobes Aerotolerant Microaerophile anaerobes s Do ox n’t u ca ygen se n its gro but pre w sen in ce.. Thioglycollate broth: Sodium thioglycollate is a reducing agent and combines with Oxygen. METHYLENE BLUE Indicator is added to the broth to show the presence of Oxygen (should only be blue in TOP of tubes Obligate Anaerobe – NOT at top. usually they won’t even go up this high Obligate Aerobe – only in top Aerotolerant anaerobe ‐ uniform growth from top to bottom. OR stringy material growth(dependent on bacteria motitlity) Facultative anaerobe – throughout tube with more growth at the top than bottom Obligate Aerobe –only in the top of the tube where oxygen is present. microaerophile (NOT shown)– vertically restricted to one zone, not at top or bottom. Thioglycollate Broth (THIO) is an enriched nonselective liquid medium used for the growth of microaerophilic and anaerobic bacteria, including fastidious organisms, from clinical specimens. Anaerobic Culture Methods Reducing media – Contain chemicals (thioglycollate or oxyrase) that combine O2 – Heated to drive off O2 – Oxygen Indicator – Methylene blue, Resazurin (pink) Thioglycollate broth: Sodium thioglycollate is a reducing agent and combines with Oxygen. METHYLENE BLUE Indicator is added to the broth to show the presence of Oxygen (should only be blue in TOP of tubes Anaerobic Culture Methods Brewer anaerobic jar – CO2 and H2 are given off when H2O is added to the envelop. – Gas-Pak with a palladium catalyst combines H2 with O2 to form H2O Figure 6.5 Physical Requirements - Temperature Temperature – Minimum growth temperature – Optimum growth temperature – Maximum growth temperature Temperature Psychrophiles are cold-loving bacteria. Their optimum growth temperature is between -5oC and 15oC. They are usually found in the Arctic and Antarctic regions and in streams fed by glaciers. Mesophiles are bacteria that grow best at moderate temperatures. Their optimum growth temperature is between 25oC and 45oC. Most bacteria are mesophilic and include common soil bacteria and bacteria that live in and on the body. Thermophiles are heat-loving bacteria. Their optimum growth temperature is between 45oC and 70oC and are commonly found in hot springs and in compost heaps. Hyperthermophiles are bacteria that grow at very high temperatures. Their optimum growth temperature is between 70oC and 110oC. They are usually members of the Archae and are found growing near hydrothermal vents at great depths in the ocean. Temperature Figure 6.1 Physical Requirements – Osmotic Pressure Osmosis - diffusion of water across a membrane from an area of higher water concentration (lower solute concentration) to lower water concentration (higher solute concentration). Osmosis is powered by the potential energy of a concentration gradient and does not require the expenditure of metabolic energy. A solution consists of a solute dissolved in a solvent. In terms of osmosis, solute refers to all the molecules or ions dissolved in the water (the solvent). When a solute such as sugar dissolves in water, it forms weak hydrogen bonds with water molecules. While free, unbound water molecules are small enough to pass through membrane pores, water molecules bound to solute are not. Therefore, the higher the solute concentration, the lower the concentration of free water molecules capable of passing through the membrane. A cell can find itself in one of three environments: isotonic, hypertonic, or hypotonic. (The prefixes iso-, hyper-, and hypo- refer to the solute concentration). Physical Requirements – Osmotic Pressure Osmotic Pressure – Hypertonic environments, increase salt or sugar, cause plasmolysis – Extreme or obligate halophiles require high osmotic pressure – Facultative halophiles tolerate high osmotic pressure Effect of tonicity on cells Physical Requirements – pH Microorganisms can be placed in one of the following groups based on their optimum pH requirements: Neutrophiles grow best at a pH range of 5 to 8. Acidophiles grow best at a pH below 5.5. Acaliphiles grow best at a pH above 8.5. The Requirements for Growth: Physical Requirements pH – Most bacteria grow between pH 6.5 and 7.5 – Molds and yeasts grow between pH 5 and 6 – Acidophiles grow in acidic environments Buffers – A solution composed of an acid and its conjugate base that serves to moderate the pH of the solution. – Acids produced by many bacteria are neutralized by buffers. Oxygen and the Growth of Bacteria Carbohydrate Metabolism Fermentation of Carbohydrates Starch Plate The culture is spread on the plate and incubated at 37oC The plates are then flooded with iodine. Iodine is an indicator that changes from amber to blue-black in the presence of starch. - Clear Zone = Starch Metabolism OF Test - To determine the oxidative or fermentative metabolism of a carbohydrate. The OF test is used to determine whether a bacterium has the enzymes necessary for the aerobic breakdown of glucose (ie oxidation) and/or for the fermentation of glucose. 1. 2. 3. Inoculate two tubes of OF medium for each organism being tested. Inoculation is carried out as a stab to within 1 cm of the bottom of the tube. Overlay one tube only with sterile paraffin oil to exclude all oxygen. Incubate at 37°C for 48 hours. OF Glucose - Semi solid agar - needle innoculation - 1 tube with mineral oil/1 not Used to determine whether an organism is oxidative or fermentative Semisolid agar deep containing a high concentration of carbohydrate and a low concentration of peptone – Peptone supports the growth of nonoxidative, non-fermentative bacteria pH indicator: bromothymol blue – Turns yellow in acidic environment Oxidative-Fermentation (OF) Media OF- Glucose semisolid agar deep The culture is inoculated into the agar in 2 tubes. A layer of mineral oil poured over the top of one. What is the purpose of the semisolid media? (What characteristic of the microbe does it allow one to observe?) What is the purpose of the mineral oil? If growth is observed in the tube with the mineral oil, what does that tell you about the action of the microbe? Growth of microorganisms in this medium is either by utilizing the peptone which results in an alkaline reaction (dark red color) or by utilizing glucose, which results in the production of acid (turning phenol red to yellow). Oxidative utilization of the carbohydrate will result in acid production (yellow) in the open tube only. Fermentative utilization of the carbohydrate will result in acid production (yellow) in both the open and closed tubes. Acidic changes in the overlaid tubes are considered to be a result of true fermentation, while acidic development in the open tubes are due to oxidative utilization of the carbohydrate present. Fermentation tube – used to detect acid and gas production from carbohydrates – contains peptone, phenol red (pH indicator), 0.5-1% of the desired carbohydrate and an inverted Durham tube to trap gas Phenol red – Red in a neutral or alkaline solution (-) – Turns yellow in the presence of acid (+) Acid production should result in a change from red to yellow, and gas production would be seen by formation of a gas bubble in the small tube. Fermentation tubes (Glucose, lactose, sucrose) MRVP Used to distinguish between organisms that produce large amounts of acid (MR) and those produce neutral product acetoin (VP). MR test: add 4-5 drops of methyl red – Red (+): acid production – Yellow (-): no acid production VP test: add 10 drops of 5% α-napthol and 10 drops of 40% KOH – Pink to red (+): acetoin production – No color change (-): no acetoin production MR Test Innoculate the broth media with the assigned culture and incubate at 37oC for 48 hours. Transfer 1ml (5 drops) of methyl red to the tube The pH range of the methyl red indicator is 4.4-6. If the indicator remains red when added to the inoculated broth this indicates that acids were produced which lowered the pH of the medium. If there is no or low production of acids or production of other neutral end products, the pH of the medium remains fairly unchanged and the indicator shows yellow color. VP (Voges Proskauer) Test Innoculate the broth media with the assigned culture and incubate at 37oC for 48 hours. Aseptically transfer 1ml of the culture to a clean test tube Add 15 drops of reagent A (5% α-napthol) followed by 10 drops of reagent B (40% KOH). During glucose fermentation, pyruvic acid is produced. It may be further metabolized through various metabolic pathways, depending on the enzyme systems possessed by different bacteria. One such pathway results in the production of acetoin (acetyl methyl carbinol), a neutral-reacting end product. If present, acetoin is converted to diacetyl in the presence of α-naphthol, strong alkali (40% KOH), and atmospheric oxygen. The diacetyl and guanidine-containing compounds found in the peptones of the broth then condense to form a pinkish-red polymer. Methyl Red test (MR) 1. Principle - To test the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Method 1. Inoculate buffered glucose broth and incubate at 37°C for 48 hours. 2. Add a few drops of methyl red solution to the culture. Read immediately. 2. 1. Special features - The test is used to differentiate between genera. eg. E. coli (+) from enterobacter (-); Protein Catabolism Motility assay – fuzzy spreading is + Decarboxylation/ MIO DEEP Ornithine decarboxylase: Ornithine Ornithine decarboxylase (amine)+ CO →Putrescine 2 (putrescine and cadaverine are very smelly amines!!! Think about names) MIO deep (motility / indole / ornithine deep) assays all of the following: 1.Motility (semi‐solid agar deep) 2.Indole production (Kovac’s reagent) Ornithine decarboxylation (bromcresol purple indicator) Ornithine is its only decarboxylat-able compound Bromcresol purple (pH indicator): Lavender / purple: alkaline = +for decarboxylation Yellow: acidic = Negative result – for this decarboxylation PROTEIN CATABOLISM – MIO DEEP (INDOLE) Some bacteria have ability to convert the amino acid trytophan to indole using tryptophanase, to indole, ammonia, and pyruvic acid. Tryptophan ammonia Tryptophanase → Indole + Pyruvic acid + Indole production can be detected by adding 4‐5 drops of Kovac’s reagent to MIO medium. 2 indole + Kovac’s reagent è Rosindole Dye Formation of bright red dye from acid condensation reaction results in a red ring on the surface of the medium indicating a (+) result Tube E is + for indole production / tryptophanase activity UREA Hydrolysis Urea is a waste product of protein digestion and excreted in the urine Urease: liberates ammonia from urea, causing dramatic pH shift Urease Urea + H O → 2 NH + CO 2 3 2 Urea broth contains peptone, glucose, urea and phenol red indicator oyellow = acidic ocopper/salmon = neutral, Negative / Uninoculated– pH 6.8 or lower ofuchsia (hot pink) = strongly alkaline, Positive result – pH 8.4 or higher Gelatin Hydrolysis Gelatin is hydrolyzed by the exoenzyme gelatinase Nutrient gelatin media has gelatin protein, which solidifies (gels) when o cooled below 20 C, and liquefies (sols) when heated. When the gelatin is hydrolyzed by gelatinase, it liquefies and does o not solidify even when cooled below 20 C. –solidifies (gels) at 4ºC : (‐) result; no hydrolysis of the gelatin, ‐ for gelatinase –liquefies (sols) at 4ºC: (+) result; hydrolysis of the gelatin occurred, + for gelatinase PROTEIN CATABOLISM – DEAMINATION Deamination is the removal of amino group from the end of the protein. Results in organic acid production Result: pH increase overall due to release of ammonia (NH ), which is a stronger 3 base than the organic acids are as acids (longer carbons chains) E.g. Phenylalanine Phenylalanine deaminase → Phenylpyruvic acid + NH 3 To detect Phenylpyruvic acid: Fe Cl : Adding 4 or 5 drops of 10% ferric chloride solution to phenylalanine slant: 2 3 dark green color indicates a ferric ion complex with the organic acid Phenylpyruvic acid + Ferric ion (Fe 3+ ) → → → Green complex To detect NH : 3 Nessler’s reagent: deep yellow indicates the presence of ammonia. NH + Nessler’s reagent → → → Yellow 3 Tube on LEFT is + for phenylalanine deamination. NOTE: phenylpyruvate may diffuse deep into agar during incubation and be detected there, rather than at the agar surface PROTEIN CATABOLISM – PEPTONE IRON AGAR DEEP / Hydrogen Sulfide Production When cysteine, cystine or methionine are metabolized (amino acids containing sulfur), you get sulfur / ammonia / organic acid production H S production can be detected by adding heavy metal salts such as iron 2 to media oPeptone iron agar deep has iron in form of ferrous sulfate When H S gas is produced, the sulfide reacts with the metal salt to 2 produce a visible black precipitate. Thus, in presense of cysteine desulfhydrase: oCysteine è H S + NH + pyruvic acid 2 3 oFerrous sulfate + H S è black precipitate (ferrous sulfide) 2 Middle and right tubes are both + for cysteine desulfhydrase. Left tube is ‐ Week 11 - pGlo See the weekly posts