Microbiology Bacteria
Phenol Red Broth
-Checks for the ability to ferment glucose, lactose, and sucrose 1. Test for the fermentation of the added carbohydrate. Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate. We use three different kinds of phenol red broths. One contains glucose; one contains lactose, and the last contains sucrose. The objective of the exercise is to determine which organisms can utilize each sugar. Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube. If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed. More in-depth information on the biochemical pathways involved in carbohydrate fermentation can be found in your lab manual.
Figure Out your unknown
-The first step is to streak your unknown broth for isolation on a TSA plate and incubate it for 24-48 hours. Be sure to vortex your broth before inoculating from it! -After incubation, look at your plate. Did you achieve separation of two distinct colonies? If so, streak each organism out on its own TSA plate and then continue with your investigation. If not, try your streak for isolation again. -When you streak a separate plate for each isolated organism, be sure to label each plate properly! You may choose to call one organism pick A and the other pick B. Whatever you decide to call them, be sure to label everything you do and keep your organisms separate! If you get confused about which organism you used to inoculate a test, you'll have to do the test all over again, and you'll lose time. Stay as organized as you can, and you'll make your life much easier. -Perform a Gram stain on your organism. Just like last time, when Gram staining an unknown, the best method is to make three bacterial smears on the same slide. One should be a known Gram positive organism like Staphylococcus aureus. The other should be a known Gram negative organism like Escherichia coli. In the middle, make a smear of your unknown organism. Then perform the Gram stain as usual. If the known Gram positive and Gram negative organisms look like they're supposed to, then you can be sure your unknown organism stained correctly. If the known organisms are not the correct colors, you know there was a problem with your staining technique and that you need to perform the stain again on a new set of smears. From the Gram stain slide, you should be able to determine the Gram specificity of your unknown and learn something about the cellular morphology. You may even be able to determine the cell arrangement of your organism. These will all be very helpful pieces of information in determining your unknown. -Based on your results so far, if it seems like it would be a useful source of information, perform a motility test on your organism using the semisolid motility media with the TTC indicator. The fermentation tests may provide useful information. A catalase test, blood agar plate, or nitrate reduction test may help narrow down the possible organisms. The IMViC tests might help.
Blood Agar
-differentiates microorganisms based on their reactions on bloods. Shows presence or absence of exoenzymes known as hemolysis that can break down hemoglobin in blood. Blood agar contains general nutrients and 5% sheep blood. It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism. Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin; these are called hemolysins. Bacteria can produce different types of hemolysins. Beta-hemolysin breaks down the red blood cells and hemoglobin completely. This leaves a clear zone around the bacterial growth. Such results are referred to as β-hemolysis (beta hemolysis). Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish color behind. This is referred to as α-hemolysis (alpha hemolysis). The greenish color is caused by the presence of biliverdin, which is a by-product of the breakdown of hemoglobin. If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur. This is called γ-hemolysis (gamma hemolysis). The hemolysins produced by streptococci perform better in an anaerobic environment. Because of this, it is standard procedure to streak a blood plate and then stab the loop into the agar to provide an area of lower oxygen concentration where the streptolysins can more effectively break down the blood cells.
Eosin Methylene Blue Agar
-the 2 dyes together-eosin and methylene blue-make the medium selective by inhibiting the growth of gram + bacteria but allowing many gram - (including coliforms) to grow (kind of like MacConkey agar) Eosin methylene blue agar (EMB) is a selective and differential medium used to isolate fecal coliforms. Eosin Y and methylene blue are pH indicator dyes which combine to form a dark purple precipitate at low pH; they also serve to inhibit the growth of most Gram positive organisms. Sucrose and lactose serve as fermentable carbohydrate sources which encourage the growth of fecal coliforms and provide a means of differentiating them. Vigorous fermenters of lactose or sucrose will produce quantities of acid sufficient to form the dark purple dye complex. The growth of these organisms will appear dark purple to black. Escherichia coli, a vigorous fermenter, often produces a green metallic sheen. Slow or weak fermenters will produce mucoid pink colonies. Normally-colored or colorless colonies indicate that the organism ferments neither lactose nor sucrose and is not a fecal coliform.
Nitrate Reduction Test
Ability for organisms to reduce nitrate (NO3) to nitrite (NO2) or further reduction to ammonia (NH3) or nitric oxide (NO) Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. Nitrate broth contains nutrients and potassium nitrate as a source of nitrate. After incubating the nitrate broth, add a dropperful of sulfanilic acid and α-naphthylamine. If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid. When sulfanilic acid is added, it will react with the nitrous acid to produce diazotized sulfanilic acid. This reacts with the α-naphthylamine to form a red-colored compound. Therefore, if the medium turns red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction. If the medium does not turn red after the addition of the reagents, it can mean that the organism was unable to reduce the nitrate, or it could mean that the organism was able to denitrify the nitrate or nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in the test. If the medium does not turn red after the addition of the nitrate reagents, add a small amount of powdered zinc. Be careful, as powdered zinc is hazardous! If the tube turns red after the addition of the zinc, it means that unreduced nitrate was present. Therefore, a red color on the second step is a negative result. The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed nitrous acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby produced reacted with the α-naphthylamine to create the red complex. If the medium does not turn red after the addition of the zinc powder, then the result is called a positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the medium, either, that means that denitrification took place and ammonia or molecular nitrogen were formed.
Urease Test
Detects the presence of the urease enzyme, which breaks down amides, producing ammoni Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme, called urease, that hydrolyzes urea to ammonia and carbon dioxide. The broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red. Phenol red turns yellow in an acidic environment and fuchsia in an alkaline environment. If the urea in the broth is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink. Many enterics can hydrolyze urea; however, only a few can degrade urea rapidly. These are known as "rapid urease-positive" organisms. Members of the genus Proteus are included among these organisms. Urea broth is formulated to test for rapid urease-positive organisms. The restrictive amount of nutrients coupled with the use of pH buffers prevent all but rapid urease-positive organisms from producing enough ammonia to turn the phenol red pink.
Staphylococcus epidermidis
Gram-positive, Cocci
Aspergillus Niger
FUNGUS used to produce citric acid for food and sodas, SOY SAUCE,septet hyphae (look like bamboo-white), conidiophores, black condo/conidiospores
Rhizopus stolonifer
Fungi. Zygomycetes. Filaments. Asexual spore.
Enterococcus Faecalis
G+ cocci; staphylo or strepto; non acid fast, non spore former, CATALASE -; grows in 6.5% NaCl in BHI (turbid)
Streptococcus bovis
Gram + , catalase - cocci, (-) in 6.5% NaCl
Bacillus megaterium
Gram + Rod, Catalase +, No pigment, Lactose -, Fermentation -, facultative anaerobe, Endospores formed in middle of rod
Mycobacterium smegmatis
Gram + rod, non-spore former, acid fast off-white, waxy appearance, pellicle with flocculent appearance in TSB -Endospore (-)
Micrococcus luteus
Gram +, Coccus, obligate aerobes, catalase +, oxidase +, Bacitracin S, catalase positive, growth on MSA, does not ferment mannitol, yellow colonies
Streptococcus pneumoniae
Gram +, Diplococci, Encapsulated. Has IgA protease.
Streptococcus pyogenes
Gram +, non motile facultative anaerobic bacterium, capsule
Rhodospirillum rubrum
Gram - sprillum, red in color, non-fermenter,
Salmonella typhimurium
Gram -, Bacilli, flagellated, - carb, - fructose, - lactose
Shigella flexneri
Gram Negative Bacillus lactose negative, acid from glucose only, no gas, urea negative, phenylalanine negative, red/yellow. gram negative rod
Enterobacter Aerogenes
Gram Negative, Bacillus, Fermenter, Negative Indole, Methyl Red tests, positive Voges- Proskauer, Citrate test
Streptococcus (Lactococcus) lactis
Gram Positive Cocci Lactic acid produce (-) catalase, (-) nitrate aerotolerant anaerobic sphere
Clostridium Sporogenes
Gram Positive rod, spore former, anaerobic
Bacillus subtilis
Gram Positive, Ednospore stain, positive for gelatinase, positive for amalayse, positive for starch
Streptococcus agalactiae
Gram+ cocci , B-hemolytic, bacitracin resistant Prokaryotic,
Proteus mirabilis
Gram-Negative Bacilli - Enteric trac "swarming" growth, urease + that can be described as oxidase-negative, nitrate-positive, indole-negative, and H2S-positive is
Enterobacter Cloacae
Gram-negative rods, Decarboxylase test lactose positive, indole negative, methyl red negative, voges proskauer positive, citrate positive, Y/Y. bubbles and cracks, gram negative rods
Triple Sugar Iron Agar
Identification of Gram negative enteric bacteria Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH indicator phenol red. It is used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates. As with the phenol red fermentation broths, if an organism can ferment any of the three sugars present in the medium, the medium will turn yellow. If an organism can only ferment dextrose, the small amount of dextrose in the medium is used by the organism within the first ten hours of incubation. After that time, the reaction that produced acid reverts in the aerobic areas of the slant, and the medium in those areas turns red, indicating alkaline conditions. The anaerobic areas of the slant, such as the butt, will not revert to an alkaline state, and they will remain yellow. This happens with Salmonella and Shigella.
Lipase Test
Identify bacteria capable of producing the exoenzyme lipase In our lab, the only lipase test we perform is the tributyrin agar test. Tributyrin agar is a differential medium that tests the ability of an organism to produce an exoenzyme, called lipase, that hydrolyzes tributyrin oil. Lipases break down lipids (fats). Tributyrin oil is a type of lipid called a triglyceride. Other lipase tests use different fat sources such as corn oil, olive oil, peanut oil, egg yolk, and soybean oil. Lipase allows the organisms that produce it to break down lipids into smaller fragments. Triglycerides are composed of glycerol and three fatty acids. These get broken apart and may be converted into a variety of end-products that can be used by the cell in energy production or other processes. Tributyrin oil forms an opaque suspension in the agar. When an organism produces lipase and breaks down the tributyrin, a clear halo surrounds the areas where the lipase-producing organism has grown.
Disinfectants
In our lab, we perform a test to determine the effectiveness of various disinfectants. This test is similar to the Kirby-Bauer test for antimicrobial susceptibility. We streak a nutrient agar plate for confluent growth and then apply to the plate sterile paper disks that we have soaked in a disinfectant. 10% bleach, 100% bleach, 10% Lysol, 100% Lysol, Listerine, isopropyl alcohol, tea tree oil, green soap, peppermint oil, and alcohol foam will be included among the disinfectants you can choose from. After incubation, zones of inhibition will be apparent around the disinfectants that are effective against the organism that has grown on the plate. Chemical products that destroy all bacteria, fungi, and viruses (but not spores) on surfaces.
Antimicrobial Susceptibility Test
Purpose is to determine susceptibility or resistance of bacteria to antimicrobial drug -n our lab, we may use either Mueller Hinton or nutrient agar plates for this test. Some instructors will have you dilute your broth culture with sterile saline to match the 0.5 McFarland standard while others will have you use the undiluted broth culture. Although the results are more striking when the test is run properly, you will still see positive results. After the plates have been inoculated, a variety of paper disks will be added. These disks have been infused with a specific amount of a microbial agent. The plates are then incubated. After incubation, there will be "bacteria-free" circles of varying sizes around some of the disks. These are called zones of inhibition. They indicate that the organism was susceptible to the antimicrobial agent. The larger the zone of inhibition surrounding an antimicrobial agent is, the more susceptible the organism is to the antimicrobic.
MacConkey Agar
Selects for gram negative organisms MacConkey Agar (MAC) is a selective and differential medium designed to isolate and differentiate enterics based on their ability to ferment lactose. Bile salts and crystal violet inhibit the growth of Gram positive organisms. Lactose provides a source of fermentable carbohydrate, allowing for differentiation. Neutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8. Organisms that ferment lactose and thereby produce an acidic environment will appear pink because of the neutral red turning red. Bile salts may also precipitate out of the media surrounding the growth of fermenters because of the change in pH. Non-fermenters will produce normally-colored or colorless colonies.
SIM MEDIUM
Sulfer Indole Production Motility used to identify bacteria that are capable of producing indole, using the enzyme tryptophanase The sulfur reduction test is useful in differentiating enteric organisms. The indole test is a component of the IMViC series of tests, which is used for differentiating the Enterobacteriaceae. The motility test is useful for testing a wide variety of organisms. As a whole, the SIM test is primarily useful for differentiating Salmonella and Shigella. SIM medium contains nutrients, iron, and sodium thiosulfate. One of the nutrients is peptone, which contains amino acids, including tryptophan. If an organism can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron to form ferric sulfide, which is a black precipitate. If there is any blackening of the medium, it indicates the reduction of sulfur and is a positive result. The sulfur and motility test results should be determined before you perform the indole test. Some bacteria possess the ability to produce the enzyme tryptophanase, which hydrolyzes tryptophan. The end products of this hydrolyzation are indole, pyruvic acid, and ammonia, by way of deamination. The Kovac's reagent that you add to the SIM medium to test for indole contains hydrochloric acid, p-dimethylaminobenzaldehyde (DMABA), and n-amyl alcohol. DMABA reacts with indole to produce a red quinoidal compound. If the reagent turns red, the indole test is positive.
Staphylococcus aureus
Yellow pigmentation Gram (+) | Cocci | Catalase (+), in clusters (Staphylococcus) | Coagulase (+) | ?
Bacitracin & Optochin Susceptibility
Test used to differentiate between Streptococcus pyogenes and Strep. pneumoniae -The bacitractin and optochin sensitivity tests identify whether an organism is susceptible or resistant to optochin and bacitracin. A blood agar plate is streaked for confluent growth with the organism to be tested, and paper disks that have been infused with either bacitracin or optochin are applied to the surface of the agar. The plate is then incubated. If the organism grows up to the edge of the disk, it is resistant to the antimicrobial compound infusing the disk. If there is a zone around the edge of the disk where the organism has not grown, the organism is susceptible to the antimicrobial in the disk. Bacitracin is a true antibiotic in that it is an antimicrobial compound which is naturally produced by a microorganism. It is produced by Bacillus lichenformis and acts to interrupt the formation of the bacterial cell wall. It is not effective on bacteria that do not have cell walls and are not actively growing. The bacitracin test is useful for differentiating β-hemolytic Group A streptococci from β-hemolytic non-Group A streptococci. This is important because most streptococcal diseases are caused by Group A streptococci. The bacitracin test can also be used to differentiate the bacitracin-resistant Staphylococcus from the bacitracin-susceptible Micrococcus.
Phenylalanine Deaminase Test
Tests for deamination of phenylalanine resulting in phenylpyruvic acid which reacts with Fe to cause green color Phenylalanine deaminase medium tests the ability of an organism to produce the enzyme deaminase. This enzyme removes the amine group from the amino acid phenylalanine and releases the amine group as free ammonia. As a result of this reaction, phenylpyruvic acid is also produced. Phenylalanine agar, also known as phenylalanine deaminase medium, contains nutrients and DL-phenylalanine. It is used to differentiate members of the genera Proteus, Morganella (which were originally classified under the genus Proteus), and Providencia from other Enterobacteriaceae. After incubation, 10% ferric chloride is added to the media; if phenylpyruvic acid was produced, it will react with the ferric chloride and turn dark green. If the medium remains a straw color, the organism is negative for phenylalanine deaminase production.
Gelatinase Test
Tests for the production of gelatinase, an enzyme that can break down the gelatin into smaller polypeptides Determine if the bacteria has gelatinous enzyme to hydrolyze the molecule (substrate) gelatin. Gelatin will liquify when hydrolyzed Nutrient gelatin is a differential medium that tests the ability of an organism to produce an exoenzyme, called gelatinase, that hydrolyzes gelatin. Gelatin is commonly known as a component of gelled salads and some desserts, but it's actually a protein derived from connective tissue. When gelatin is at a temperature below 32°C (or within a few degrees thereof), it is a semisolid material. At temperatures above 32°C, it is a viscous liquid. Gelatinase allows the organisms that produce it to break down gelatin into smaller polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized by the organism. When gelatin is broken down, it can no longer solidify. If an organism can break down gelatin, the areas where the organism has grown will remain liquid even if the gelatin is refrigerated.
Coagulase Test
The coagulase test is useful for differentiating potentially pathogenic Staphylococci such as Staphylococcus aureus from other Gram positive, catalase-positive cocci. The coagulase test identifies whether an organism produces the exoenzyme coagulase, which causes the fibrin of blood plasma to clot. Organisms that produce catalase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, and some other antimicrobial agents. The coagulase slide test is used to identify the presence of bound coagulase or clumping factor, which is attached to the cell walls of the bacteria. Bound coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate. This causes the cells to agglutinate, or clump together, which creates the "lumpy" look of a positive coagulase slide test. You may need to place the slide over a light box to observe the clumping of cells in the plasma. The coagulase tube test has been set up as a demo for you to observe in class. This version of the coagulase test is used to identify the presence of either bound coagulase or free coagulase, which is an extracellular enzyme. Free coagulase reacts with a component of plasma called coagulase-reacting factor. The result is to cause the plasma to coagulate. In the demo, the coagulase plasma has been inoculated with Staphylococcus aureus and Staphylococcus epidermidis and allowed to incubate at 37˚C for 24 hours. Staphylococcus aureus produces free coagulase; Staphylococcus epidermidis does not.
6.5% salt tolerance test
The salt tolerance test is performed using Tryptic Soy Broth with added sodium chloride (regular table salt) to create an overall salt concentration of 6.5%. It is a selective medium which tests the ability of an organism to survive in a salt-rich environment. Most organisms cannot survive in such an environment. Staphylococci, Enterococci,and Aerococci are expected to grow in this broth; other organisms are not.
Escherichia Coli
What facultative gram-negative anaerobic rod is motile, ferments lactose, and is the MCC of UTIs? Form bright pink-red colonies
Hektoen Enteric Agar
a selective and differential media used to isolate and distinguish between Salmonella and Shigella species Hektoen Enteric Agar (HE) is a selective and differential medium designed to isolate and differentiate members of the species Salmonella and Shigella from other Enterobacteriaceae. Bile salts and the dyes bromthymol blue and acid fuchsin inihibit the growth of most Gram positive organisms. Lactose, sucrose, and salicin provide fermentable carbohydrates to encourage the growth and differentiation of enterics. Sodium thiosulfate provides a source of sulfur. Ferric ammonium citrate provides a source of iron to allow production of hydrogen sulfide from sodium thiosulfate, which provides a source of sulfur. Ferric ammonium citrate also allows the visualiztion of hydrogen sulfide production by reacting with hydrogen sulfide gas to form a black precipitate. Enterics that ferment one or more of the carbohydrates will produce yellow to salmon-colored colonies. Non-fermenters will produce blue-green colonies. Organisms that reduce sulfur to hydrogen sulfide will produce black colonies or blue-green colonies with a black center.
Citrate Test (simmons citrate agar test)
ability of an organism to use citrate as its sole carbon source (instead of utilizing glucose) Blue dye will show positive blue color when pyruvate produced and increase in pH Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. Simmons citrate agar contains sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen, other nutrients, and the pH indicator bromthymol blue. This test is part of the IMViC tests and is helpful in differentiating the Enterobacteriaceae . Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to transport the citrate into the cell. These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an alkaline environment in the medium. At pH 7.5 or above, bromthymol blue turns royal blue. At a neutral pH, bromthymol blue is green, as evidenced by the uninoculated media. If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative. Some citrate negative organisms may grow weakly on the surface of the slant, but they will not produce a color change.
Catalase Test
biochemical test differentiates Streptococcus species from Staphylococcus species. -Gram positive cocci and small bacilli What tests can you use to distinguish Staph from Strep?Catalase is the enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2. Hydrogen peroxide is often used as a topical disinfectant in wounds, and the bubbling that is seen is due to the evolution of O2 gas. H2O2 is a potent oxidizing agent that can wreak havoc in a cell; because of this, any cell that uses O2 or can live in the presence of O2 must have a way to get rid of the peroxide. One of those ways is to make catalase.
Decarboxylation Test
differentiate organisms in Enterobacteriaceae and distinguish them from other Gram- rods - tests for the production of the enzyme decarboxylase, which removes the carboxyl group from an amino aci Decarboxylase broth tests for the production of the enzyme decarboxylase, which removes the carboxyl group from an amino acid. Decarboxylase broth contains nutrients, dextrose (a fermentable carbohydrate), pyridoxal (an enzyme cofactor for decarboxylase), and the pH indicators bromcresol purple and cresol red. Bromcresol purple turns purple at an alkaline pH and turns yellow at an acidic pH. We also add a single amino acid to each batch of decarboxylase broth. The three amino acids we test in our decarboxylase media are arginine, lysine, and ornithine. The decarboxylase test is useful for differentiating the Enterobacteriaceae. Each decarboxylase enzyme produced by an organism is specific to the amino acid on which it acts. Therefore, we test the ability of organisms to produce arginine decarboxylase, lysine decarboxylase, and ornithine decarboxylase using three different but very similar media. If the organism is unable to ferment dextrose, there will be no color change in the medium. If an organism is able to ferment the dextrose, acidic byproducts are formed, and the media turns yellow. As the organisms ferment the dextrose, the media initially turns yellow, even when it has been inoculated with a decarboxylase-positive organism. The low pH and the presence of the amino acid will cause the organism to begin decarboxylation. If an organism is able to decarboxylate the amino acid present in the medium, alkaline byproducts are then produced. Arginine is hydrolyzed to ornithine and is then decarboxylated. Ornithine decarboxylation yields putrescine. Lysine decarboxylation results in cadaverine. These byproducts are sufficient to raise the pH of the media so that the broth turns purple. If the inoculated medium is yellow, or if there is no color change, the organism is decarboxylase-negative for that amino acid. If the medium turns purple, the organism is decarboxylase-positive for that amino acid.
Alcaligenes Faecalis
gram - NON fermenter, TSI K/K, bacilli, non saccharolytic O/F media both green
Proteus vulgaris
gram - bacillu positive for urease organism most likely growing in tubes (+) for cysteine desulfurase
Serratia marcescens
gram - rod, middle growth in Fluid Thioglycollate Medium, red, Lactose + GNB, H2S: neg, sucrose: neg, VP: pos, citrate: pos, LDC: pos, gelatin: pos, may produce a red pigmentation
Neisseria sicca
gram negative diplococc, + oxidase
Neisseria subflava
gram negative, cocci, + catalase, + oxidase
Bacillus cereus
gram positive, aerobic, spore formin, non encapsulated, motile
Klebsiella Pneumoniae
gram-negative, Bacilli, encapsulated, lactose-fermenting
Pseudomonas aeruginosa
gram-negative, oxidase-positive aerobic rod that produces a grapelike odor and pyocyanin pigmentation?
DNase Test
identifies bacteria that produces DNase exoenzyme such as staphylococcus aureus, streptococcus pyogenes, and serratia marcesscens DNase agar is a differential medium that tests the ability of an organism to produce an exoenzyme, called deoxyribonuclease or DNase, that hydrolyzes DNA. DNase agar contains nutrients for the bacteria, DNA, and methyl green as an indicator. Methyl green is a cation which binds to the negatively-charged DNA. Deoxyribonuclease allows the organisms that produce it to break down DNA into smaller fragments. When the DNA is broken down, it no longer binds to the methyl green, and a clear halo will appear around the areas where the DNase-producing organism has grown.
Bile Esculin Test
inhibits growth of all gram positive bacteri selective and differential medium which is used to presumptively identify enterococci and group D streptococci based on the ability of an organism to hydrolyze esculin. - contains oxgall (bile salts) to inhibit the growth of gram positive organisms. -Bile esculin agar (we do not use bile esculin azide agar, as sodium azide is extremely hazardous) is a selective and differential medium which is used to presumptively identify enterococci and group D streptococci based on the ability of an organism to hydrolyze esculin. Bile esculin agar contains oxgall (bile salts) to inhibit the growth of gram positive organisms other than enterococci and group D streptococci. It also contains nutrients, esculin, and ferric citrate. When an organism hydrolyzes the glycoside esculin to form esculetin and dextrose, the esculetin reacts with the ferric citrate to produce a dark brown or black phenolic iron complex. If an organism can hydrolyze esculin, the media will turn dark brown or black. However, the test is interpreted as a positive result only if more than half the medium is dark brown or black after incubation.
Mannitol Salt Agar (MSA)
is a selective and differential media for detecting Gram positive bacteria Mannitol Salt Agar (MSA) is a selective and differential medium. The high concentration of salt (7.5%) selects for members of the genus Staphylococcus, since they can tolerate high saline levels. Organisms from other genera may grow, but they typically grow very weakly. MSA also contains the sugar mannitol and the pH indicator phenol red. If an organism can ferment mannitol, an acidic byproduct is formed that will cause the phenol red in the agar to turn yellow. Most pathogenic staphylococci, such as Staphylococcus aureus, will ferment mannitol. Most non-pathogenic staphylococci will not ferment mannitol.
Motility Test
members of this family possessing flagella can demonstrate motility determines production of flagella; tests turbidity; positive results show cloudiness away from stab ling; negative show growth only along stab line When you're ready to read your inoculated tube, carefully hold it up to the light. It helps to hold an uninoculated tube next to it for comparison. Is the inoculated tube cloudy at all? Is it more pink? Or is there a pink streak (the stab line) with crisp edges? If the needle didn't go straight in and out, the stab line may not be a simple up-and-down line. If the needle was bent or your hand shook, the needle may have cut through the agar. Typically, though, such a cut only happens in one direction. If the growth of your organism spreads out in only one direction, and if there is a sharply differentiated edge between this growth and the agar, your organism is nonmotile. It has spread side-to-side only because those are places where the needle contacted the agar; in other words, the organism only grew where it was inoculated. If the growth around the stab line has radiated outwards in all directions (it will often look fuzzy) or if the entire tube has turned pink, your organism is motile.
Kocuria rosea (Micrococcus roseus)
pink colonies, catalase +, gram positive cocci, non mannitol fermenter, translucent TSA growth
Penicillium notatum
produces penicillin, conidiospores, formed in chains -no gram stain/shap
Phenylethyl Alcohol Agar
selective for G+ bacteria 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.
Mr-VP tests
test differentiates between mixed acid and butanediolh fermenters. Methyl Red (MR) and Voges-Proskauer (VP) broth is used as a part of the IMViC tests as the medium in which both the Methyl Red and Voges-Prosakuer tests can be performed. It is a simple broth that contains peptone, buffers, and dextrose or glucose. Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways. Some of these pathways produce unstable acidic products which quickly convert to neutral compounds. Some organisms use the butylene glycol pathway, which produces neutral end products, including acetoin and 2,3-butanediol. Other organisms use the mixed acid pathway, which produces acidic end products such as lactic, acetic, and formic acid. These acidic end products are stable and will remain acidic. The Methyl Red test involves adding the pH indicator methyl red to an inoculated tube of MR-VP broth. If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium. When methyl red is added, if acidic end products are present, the methyl red will stay red. NOTE: Methyl red differs from Phenol red (which is used in the fermentation test and the MSA plates) in that it is yellow at pH 6.2 and above and red at pH 4.4 and below. Phenol red turns yellow below a pH of 6.8. If you get these two pH indicators confused, you will have a difficult time interpreting test results. The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result. If, after the reagents have been added, a copper color is present, the result is negative. The MR and VP tests are particularly useful in the identification of the Enterobacteriaceae.
Casease Test
test for the enzyme casease which hydrolyze casein (protein in milk). Positve is when the plate begans to lose the whiteness and negative is when nothing happens. Skim milk agar is a differential medium that tests the ability of an organism to produce an exoenzyme, called casease, that hydrolyzes casein. Casein forms an opaque suspension in milk that makes the milk appear white. Casease allows the organisms that produce it to break down casein into smaller polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized by the organism. When casein is broken down into these component molecules, it is no longer white. If an organism can break down casein, a clear halo will appear around the areas where the organism has grown.
Oxidase Test
used to differentiate the genera Neisseria and Moraxella (positive) from Actinobacter (negative); following the addition of the oxidase reagant, the colony will turn bright blue/purple -The oxidase test identifies organisms that produce the enzyme cytochrome oxidase. Cytochrome oxidase participates in the electron transport chain by transferring electrons from a donor molecule to oxygen. The oxidase reagent contains a chromogenic reducing agent, which is a compound that changes color when it becomes oxidized. If the test organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds.
Starch Hydrolysis
uses a starch agar to test for starch hydrolysis, after plate is streaked and incubated it is flooded with Gram's iodine Starch agar is a differential medium that tests the ability of an organism to produce certain exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch. Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism. Starch agar is a simple nutritive medium with starch added. Since no color change occurs in the medium when organisms hydrolyze starch, we add iodine to the plate after incubation. Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence of starch. A clearing around the bacterial growth indicates that the organism has hydrolyzed starch.
