do a 300 words minimum lab report.

do a 300 words minimum lab report.
35 Sulfur / Indole / Motility deeps, i.e. SIM deeps SIM deeps: SIM is a medium, NOT a test. Actually, we can conduct 3 tests with this single medium: 1. sulfur reduction 2. release of indole from utilization of Tryptophan and 3. motility We will conduct the Indole test as well as the sulfur reduction (H2S production) test using SIM deeps. Many medical labs use SIM deeps (Sulfur Indole Motility) in conjunction with TSI, urea and Simmons citrate media for characterization and identification of enteric bacteria. To inoculate, stab 1 SIM deep using your needle exactly as you did when inoculating a motility deep in one of our previous exercises. Incubate for 24-48hrs. at 37oC. Add 3-5 drops of Kovacs reagent and set aside for 5 minutes. Examine the top layer of the tube for red color which indicates a positive Indole test. Sulfur reduction: As discussed in the TSI exercise, the ability to generate hydrogen sulfide (H2S) can be used to distinguish the enteric genera. Thiosulfate is present in SIM media which is reduced to hydrogen sulfide (H2S) during anaerobic respiration. H2S combines with Ferric iron in this medium, and precipitates as black FeS. The black compound is obvious in an incubated tube. Sulfur negative: Enterobacter, E. coli, Klebsiella, Shigella, and all the non-fermentors positive: Proteus, Salmonella, Citrobacter SEE IMAGE: Sulfur reduction: left positive, right negative Indole: Some bacteria are capable of using the amino acid tryptophan as a carbon and energy source during the process of aerobic respiration, releasing indole as a by-product. Being produced primarily at the top of the tube, as well as accumulating there due to its non-polar nature, indole will be concentrated on the top of the media where it can be detected by Kovac’s reagent (paradimethylamino-benzaldehyde in HCl and amyl alcohol). Before using the tryptophan, bacteria split it into 3 parts: a) indole (see structure) b) an amine group -NH2 c) pyruvate which is respired by the cell If a reagent such as Kovac’s is added to indole, a thin layer of red color will develop at the top of the deep indicating the presence of indole. The color may become obvious in a few seconds, or it may take a minute or 2. Of our cultures, only E. coli and Proteus are indole positive. Indole negative: Enterobacter, Klebsiella, Salmonella, Shigella, Citrobacter and all the non-fermentors positive: Proteus, E. coli SEE IMAGE: Indole production from tryptophan utilization: left negative, right positive Motility: As said above, SIM is a soft agar medium, and thus, a motility medium. Our non-motile Gram negative rods include Shigella, Klebsiella and Acinetobacter. Motility negative: Shigella, Klebsiella, Acinetobacter positive: Enterobacter, E.coli, Salmonella, Citrobacter, Aeromonas, Alcaligenes, Pseudomonas SEE IMAGE: Motility: left non-motile, right motile The tests described above for the characterization of enteric Gram negative rods represent only a few of the options available. Additional selective & differential media exist including: Hektoen enteric agar (HE), Salmonella Shigella agar (SS), Leifson’s deoxycholate-citrate agar (DCA), Wilson and Blair’s brilliant-green bismuth sullphite agar (BBSA) or simply brilliant green agar, Xylose lysine deoxycholate agar (XLD), Kligler’s iron agar (KIA), and bile esculin agar. Additional biochemical tests include Lysine and Ornithine decarboxylation, Urea hydrolysis, orthonitrophenyl-b -D-galactopyranoside (ONPG) for beta-galactosidase production, phenylalanine deaminase, lysine decarboxylase and other amino acid modification, nitrate reduction, denitrification, and fermentation characteristics based upon various carbohydrates. NOTES: * Other than the use of MR of differentiate Listeria (MR+) from Corynebacterium (MR-), we will only use these tests for Gram negative rods. * All of the non-enteric Gram negative rods are negative for all of these tests except for motility. * Remember that the initial weak red color for MR on Klebsiella is a false positive – the color will fade over time (somewhere between a few minutes to 24 hours). * Use Kovac’s reagent with caution. If Kovac’s reagent comes in contact with your skin wash immediately and liberally with soap and water. * Positive sulfur results mask motility results in SIM deeps. This is not an issue for us due to the fact that our only non-motile Gram negative rods (Klebsiella, Shigella and Acinetobacter) are also sulfur negative.
do a 300 words minimum lab report.
Additional methods used to characterize enteric bacteria Many species of medically important enteric Gram negative bacteria exist. Furthermore, most have similar cellular and colonial morphologies making identification difficult. For this reason, much emphasis has been placed on characterization and identification of enteric species. More than any other group of bacteria, enterics are characterized and identified on the basis of carbohydrate utilization (what sugars they eat and how they eat them) and other biochemical characteristics. We have already discussed some commonly used methods of characterizing enteric bacteria: 1) the oxidase test distinguishes between 2 families of Gram negative rods 2) MacConkey agar determines the ability (or inability) to ferment lactose 3) EMB agar does the same as MacConkey, but quantitates relative amount of acid produced 4) Simmon’s citrate agar determines the ability to utilize citric acid 5) carbohydrate fermentation broth characterizes fermentation profiles (growth, acid, gas) 6) TSI looks at 3 carbohydates at once, as well as H2S production, and peptone utilization, all in 1 tube! Most labs use 1 or more of the methods summarized above. Now lets look at some additional methods used to characterize enterics, and some other Gram negative rods as well. A group of tests used for years (and still used in some labs) to characterize enterics is known as the “IMViC” series. IMViC is an acronym which stands for Indole, Methyl red, Vogues Proskauer, and Citrate. The IMVIC series is NOT a single medium. We will conduct the Methyl red test using MR/VP broth, the Indole test using SIM deeps, and the sulfur reduction test (H2S production) also using SIM deeps. Methyl red / Vogues Proskauer (MR/VP): Some enteric bacteria can be separated into 2 groups based upon the end-products of glucose fermentation. One group, which contains the genera Escherichia, Proteus, Salmonella, Shigella and Citrobacter, conduct a mixed-acid-type fermentation meaning that they produce a variety of acidic fermentation products. A mixed-acid fermentation equates to a methyl red positive result, and a Vogues Proskauer negative result (MR+/VP-). The genera Klebsiella and Enterobacter produce a pH neutral fermentation product called butanediol, which equates to a methyl red negative result, and a Vogues Proskauer positive result (MR-/VP+). Non-enteric Gram negative rods (the Gram negative non-fermentors) will give negative results on both MR & VP reactions due to the fact that they do not ferment glucose. Although this test is used primarily for enteric Gram negative rods, it can also be used to distinguish the Gram positives rods Listeria (MR+) from Corynebacterium (MR-). MR/VP broth is a defined medium containing glucose as a source of carbon and energy. To conduct the test, inoculate 1 tube of MR/VP broth with the organism as you would inoculate a tube of carbohydrate fermentation broth, and incubate at 37oC for 24 hours. If you want to conduct the MR and the VP tests, pour half of the broth into a second clean tube following the incubation period. For the MR test, add 3 or 4 drops of methyl red reagent to the broth and swirl. If the organism is a mixed-acid fermenter (MR+), the broth will turn red in color due to the acidic pH. The red color will probably develop immediately, but may take 5 or 10 minutes. NOTE: Klebsiella will often give an apparently weak MR positive reaction immediately upon adding the MR reagent but the orange-red color will fade over time (a few minutes to several hours). This is a false positive reaction. SEE IMAGE: Methyl red: left to right: positive, weak positive, negative MR negative: Klebsiella (watch out! see NOTES), Enterobacter MR positive: Escherichia, Proteus, Salmonella, Shigella, Citrobacter For the VP test, add 4-6 drops of VP “reagent A” (potassium hydroxide). Thump the tube for ~10 seconds to mix the reagent with the culture. Next add 2 drops of VP “reagent B” (alpha napthol). Cover the tube with a piece of parafilm or something else to prevent spillage. Now shake this tube vigorously to oxygenate the solution. Shake for at least 1 minute. Set the tube in the rack and wait 15 minutes. The development of red color in the tube indicates a positive VP reaction. Of our 6 enteric organisms only E. aerogenes and K. pneumoniae are MR-/VP+. The remaining 5 are MR+/VP-. Take caution while shaking as the VP reagents cause skin irritation, and can damage clothing. For our purposes, there is no need to run the VP test. All of our enterics that are MR + are VP- and vice versa. This is good considering the fact that VP reactions are not terribly consistent and the VP reagents are unpleasant to work with.
do a 300 words minimum lab report.
31 Acid and gas production during carbohydrate fermentation Enteric species, as well as other groups of Gram negative and Gram positive bacteria, can be characterized in 3 ways that describe how they ferment a particular carbohydrate: 1. Does the organism grow? (ie. can it utilize the carbohydrate present?) If the answer to #1 is yes……… 2. Does the organism produce an acidic by-product from fermentation of the carbohydrate? 3. Does the organism produce gas from fermentation of the carbohydrate? Growth: Carbohydrate fermentation media is in the form of broth. If an organism is capable of utilizing the carbohydrate, the media will become turbid. If turbidity is not obvious following the incubation period, swirl the tube to make sure that cells have not grown and settled to the bottom of the tube. Cells of some bacterial species (such as some Bacillus species) will precipitate out of the media and form a pellet at the bottom. You could incorrectly assume that growth did not occur in this case. Acid production: As mentioned in earlier labs, the process of fermentation is characterized by incomplete digestion of some catabolite, usually a carbohydrate. Instead of totally mineralizing the catabolite, as happens in the process of respiration, fermentors only partially break down the food resulting in the formation of reduced by-products such as acids, alcohols, and other solvents. Some organisms produce acidic by-products upon fermenting a particular carbohydrate, whereas others produce neutral pH or alkaline by-products. Carbohydrate fermentation media contains the pH indicator phenol red, which turns from red at neutral pH to yellow at acidic pH. The rule of thumb is this; you should score a test positive for acid production ONLY if the media turns totally yellow from top to bottom. Some organisms will cause a weak acid reaction (yellowish-red color) throughout the tube, whereas other organisms will form a yellow color only at the top or bottom of the tube. These are considered false positive reactions. Gas production: Carbohydrate fermentation tubes also contain a small inverted vial called a Durham tube. Some fermenting organisms produce gas in addition to reduced by-products when fermenting certain carbohydrates. If the organism produces gas, bubbles will be trapped in the inverted tube for you to see. Although most manuals suggest that the presence of any amount of gas should be considered a positive reaction for gas production, a tiny bubble most likely had nothing to do with gas production by the bacteria. You will be able to tell the difference. When inoculating fermentation tubes, you must do so gently so you do not accidentally force bubbles in the Durham tube and cause a false positive. If carbohydrate fermentation results were very dependable (which, I am afraid, they are not) we could use the method to differentiate several of our enteric organisms. See the table of expected results below which reflects that seen in many manuals. For example, using lactose alone, notice how we could differentiate: E. coli from Proteus from Klebsiella & Enterobacter from Salmonella & Shigella. Each of these 4 groups gives a different profile based upon growth / acid / gas results. Notice the lactose profile for E. coli. E. coli is theoretically the only organism on our list to give positive results for growth, acid and gas on lactose (but this does not work in practice, at least in our lab). For this reason, we say the E. coli is a fecal coliform, which is defined as a Gram negative, oxidase negative rod that produces acid and gas from lactose, and is bile resistant. E. coli is the representative of the fecal coliforms. E. coli is used as the indicator organism for fecal contamination in treated wastewater, drinking water, and the food industry. Inoculate each organism that you choose to use into 1 fermentation tube of each carbohydrate type. Use a very small amount of cells as an inoculum. If you add too many cells, the media will be turbid from the beginning and you will not be able to determine for sure whether the organism grew in the media or not. Incubate the tubes at 37oC for 24-48 hours. Examine and score each tube for growth, acid production and gas production. FOR UNKNOWNS the most useful results for separating enteric genera include: 1. Acid production from sucrose: Citrobacter + vs Salmonella – 2. Gas production from glucose: Shigella – vs all other enterics + SEE IMAGES: Carbohydrate fermentation: left to right: positive acid and gas, negative acid and gas Carbohydrate fermentation: left to right: positive acid and negative gas, negative acid and gas Expected results – DO NOT trust these results E. coli Proteus vulgaris K. pneumoniae E. aerogenes Growth + Acid + Gas + +/- ↑ S. enteritidis S. flexneri Growth Acid Gas G = glucose L = lactose S = sucrose NOTES: *We will only use this test to differentiate Gram negative rods. * Did you notice the results for E. coli, the “fecal coliform,” on lactose in the table above? Remember why we say that E. coli is a fecal coliform.