Please look over this document (attached) and then fill out the chemical tests to tell me what kind of bacteria you were growing in the lab

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Please look over this document (attached) and then fill out the chemical tests to tell me what kind of bacteria you were growing in the lab. You will need to use your lab manual (posted here as a pdf) to interpret the tests that are shown in the images of the grid. You only need the links listed below if you do not understand the chemistry of the manual. Please let me know if you need the links

You will need to determine the identity of your unknown from the list provided in the file.

EACH chemical te st needs to be DESCRIBED. Not just "positive" or "negative" but what the results mean - what chemistry does that positive or negative represent? For example, let's say you have a Phenol Red Lactose tube which turns yellow and makes a bubble. This positive result means that the bacteria can eat lactose and also produced CO2.

Fill out the columns for "Appearance After" and "Interpretation" then answer the questions about gram status, endospore presence, and description of quadrant streak colonies. Once you have all that information, you should be able to use the internet to determine if you have Planococcus, Micrococcus, Escherichia, or Serratia.

 

Unknown letter C Gram Stain Endospore Stain Quadrant Streak Test Initial Appearance Methyl Red Yellow transparent & Voges– liquid Final Appearance 1- After MR Proskauer After VP Interpretation Nitrate Yellow transparent liquid No zinc added Simmons Dark green Citrate tryptone Transparent lightyellow liquid Sulfur reduction (SIM) Cloudy light yellow KIA (Kliger’s Iron Agar) Orange agar Phenylalanine Cloudy light strawcolored liquid Phenol Red Liquid hot pink Lactose Phenol Red Liquid, transparent, Mannitol hot pink Phenol Red Liquid, hot pink Dextrose Litmus Milk Lavender opaque Catalase test Upon addition hydrogen peroxide bubbling seen Was your bacteria gram positive or gram negative? Does your bacteria have endospores? Does your bacteria create a color on the quadrant streak plate? After filling out the chart shown above for columns appearance after and interpretation, which of the following bacteria do you think you have: Planococcus, Escherichia, Micrococcus, or Serratia Give me three sentences about why you believe you have what you have. • LAB 26 Pr oj ec t-I de nt ify ing an Unknown Bacterium ; \~•. _, -: ? ,-rt ·'')J'II'._, ~ (. t '~ i '"·r · · . ·~- .~ ,v ·. .., . . • ' .. . ~, .,i. Intro duc tion to the Proj ect This is a three week project where the focus of each lab will be to gather information about an unknow n bacterial culture with the ultimate goal of discovering ~.-.. .,.. y4_ ,., • • ..,'H . , what it is. You will not need to identify the bacteria f to the species-level. Identification of the Genus of e . 4: '\ ~, your unknow n bacteria will require information about its shape, what it likes to eat, how it reacts • 1'-~ ).: ': to oxygen , and its other chemical capabilities. ! . \.. .. Remem ber that describing bacteria isn't easy! Since they are all so small, a simple identification of a "rod" or a "coccu s" shape only gives the most general information. The same holds true for the .. determination of "Gram -positi ve" or "Gram 11..........__ _ _ _ _ ____....!.._:_i negative" status. In order to correctly identify the bacteria in the unknow n slant you'll need to delve deeper into its physiology. The following sections and descriptions should aid your information gathering efforts. Only once you've collected many pieces of inform ation will identifi cation be possible. Docum enting each step of your process is unnecessary but documenting your results is imperative. The ultima te identification in the 3 rd week of lab will be done using Bergey's Manual of Bacteriology. This is an encyclopedia of inform ation of many genera of bacteria and has been used for decades as the author ity in commo n bacterial descriptions. You and your lab partne r will need to be very careful about contamination of your work. Just because you don't know the identity of your bacteri a doesn' t mean that no one knows it. Most of the unknowns are opport unistic pathog ens of some kind; none have been chosen that are particu larly virulent, though of course , you must exercise caution , follow safety rules as usual, etc. There is a different kjnd of risk here; you will be workin g with your culture and tubes for several weeks. If you contam inate any of the tubes or plates with environ mental bacteria no one will know that your work is contam jnated, and no one will know what it is contam inated by! The potenti al exists to grow something infectio us or potentially lethal. In the least harmfu l situatio n, contam ination will create confusion during your attempts at identification; in the most harmful scenario, someo ne could be expose d to a serious pathog en. I, ., ,., . r'LE:~ ..,EBE CAUT,UUS AND f'(f PECTFUL OF THE WORK A D cu• IR s YOU ARE ( \ NC. OJ ARE D ING 147 I De scr ibin g Ba ct9 ria .. Mor pho logy mine as much infor mati on as po-;s iblc abo ut the The first step in identification should be to deter o r if it is to know 1f the shape is a rod, cocc us, or spira l, bacte rium's shap e and struc tures. You will need be its uld sho r different condition s. The next disco very plcomorphic- meaning the shap e cha nges unde undt: r ge-; Gram -vari able; mean ing the Gram stain chan Gram c;tarus: Gram -pos itive, Gram-negative, or thou gh nce of endo spor es will be very impo rtant . Even different conditions. Finally, the presence or abse capa ble are that eria bact of there are only a few gene ra the staining procedure for endospores is tedio us, identities. ntial pote give you a cons idera bly sho rter list of of maki ng these structures, so their prese nce will you h can be relevant for identifyin g bact eria, whic There are also othe r physical desc riptio ns that ar pink , . rf the bacteria have pigm ent, meaning they appe can o bserve by grow ing them on an agar plate h wort is eria almost colorless, this desc ripti on of bact yellow, purple, brigh t white, or even if they are are shapes when grow ing on a plate . Som e colo nies noting, as is the appe aran ce of individual colo ny dery, , rhizo id, flat, roun ded or mou nded , fu zzy, pow perfectly roun d, othe rs appe ar irregular, wrin kled onat e. uniform, bran chin g, drop -like, conv ex, or umb ribe desc to s word Noti ce Figure 26.1 has Elevation the heig ht of the colo nies (elevation), word s to and gin), (mar ies colon the of descr ibe the edge = Flat Plateau Umbonate Convex word s that describe the entir e colo ny shap e (configuration). In orde r to gather this -=.. cG>, C>, w Flat, raised . .PIJi!IS infor mati on you will need an NA plate that Raised, Raised Grow th into margin spreading has been strea ked for isola tion. Refer back to mediu m edge the quad rant strea k lab if you have forgo tten the steps . You will also need these indiv idua l Marg in colo nies to dem onst rate that you are grow ing an unco ntam inate d cultu re and to use one colo ny each to inoc ulate the basic phys iolog ical Filamentous Lobat e Irregular Rhizoid tests we will be start ing next week. (12 in all) Smoo th, ) are (erose and agar the entire into Som e bacte ria grow subs eque ntly diffi cult to pick. Othe r colo nies Whol e colon y ed are stick y in appe aran ce or strin gy whe n pick e ......... ";e.' with a loop , a qual ity know n as mucoid. Thes . _- >;.r in ful help are that s are othe r char acter istic I ,R n whe rded reco be Rhizoid ld entou s lar shou Filam Irregu and d Roun tion iden tifica for s riou noto are ies notic ed. Bacillus spec colon y featu res. FIGURE 26.1 A samp ling of bact erial emb eddi ng into the agar, and Pseu dom onas ial morp holo gy. colon ribe desc Thes~ te_rms are used to ce surfa , color aeruginosa is a typical muc oid. de inclu Descriptions also shou ld that lab buty rousfirst {dry, your cy in isten steps Desc ribe the characteristics {dull or shiny), cons or que {opa your s t ertie buttery, or moist) and optic al prop you will unde rtake to gath er data abou translucent). unkn own . DO NOT FOR GET to reco rd the lette r o f 0 O@ your unkn own tube ! _ . __ 1 J 2 3 Oxygen Requirements capabilities th at a llow t hem to have flexible metabolic Bacteria . . · survive. unusual or changmg conditions· There are certam · t · flexibili·ty have not do however, that bacteria, m erms o f t he1r . reqmrement for oxygen. Some . bacteria requi·re oxygen for growth. They are called obligate aerobes. There are other bacteria that have the preference to grow in the presence of oxygen, but can grow adequately without it. These bacteria are known as facultative anaerobes. There are also bacteria that are outright killed by exposure to oxygen, known as obligate anaerobes, but we will not be using them in class. Growing bacteria on a plate does not help us to discern bacterial oxygen requirements. We merely see growth, with no ability to dis~inguish if the bacteria have the option to grow without it. When we grow bacteria in agar slants or agar deeps, we are able to discern more information. FIGURE 26.2 Growth of a facultative anaerobe and obligate aerobe. Notice determine When inoculating any slant or deep, it will help growth throughout the tube on left the oxygen requirements of your bacteria if you also stab the (pinkish) and no growth where the agar agar with your inoculating loop, in addition to dragging the was cut on the right. bacteria on the surface. This will introduce bacteria throughout the entire length of the tube, and if the bacteria can grow, they will grow. The agar in the tube creates an oxygen gradient. The top of the tube has the most oxygen since it is the closest to the atmosphere. There is less oxygen in the middle of the tube and the bottom of the tube is anoxic. A facultative anaerobe will be able to gr;w throughout the tube, and an obligate aerobe will be found only in or near the top of the tube. Every solid agar tube should be stabbed so that if one tube doesn't grow, the others will provide confirmation of oxygen requirements. Sugar Requirements Many bacteria have the ability to live on different energy sources, though some bacteria are limited. In order to determine the flexibility of your organism there are some simple tests that can be done with different kinds of sugars. We've already seen the phenol red lactose test when we studied the contaminants of water, but there are other phenol red sugar tests that we can do. Phenol red is a pH indicator. It becomes yellow when acidic and bright pink when basic. If the bacteria are able to "eat" the sugar, it will produce acid as a by-product. This drops the pH of the tube to an acidic level and the tube changes color to yellow. If the bacteria are NOT ABLE to eat a particular kind of sugar, then they will either not grow and not change the appearance of the tube, or they will grow on the peptones in the tube and create a basic product, which will change the color to hot pink. Additionally, these tests have Durham tubes inside them to catch gas if it is produced. Common sugars in these tests include dextrose or glucose, mannose or mannitol, and lactose. We will test three sugar sources in phenol red tubes. Lab 26 Project-Identifying an Unknown Bacterium 149 r l Describing Other ... Physiological Capab1ht1e~ Below are the descriptions of 10 ~~sts that will . n re metabolic capab1ht1es of the . detcrmine 10 unknown bacteria. In real identification scenarios many more tests might be used. They would be grouped into primary tests whose results would determine some secondary tests, and the results of the secondary tests might determine a third round of tertiary tests. The testing "rounds" would continue until a positive identification to the species and strain levels was confirmed. This can take 3-5 days due to the time needed for the tests to grow. More rapid results are possible now when using PCR and sequencing, or other modern technology. 26.3 Phenol red sugar tubes: acidic with gas production, uninoculated, and negative for acid but FIGURE positive for gas. ; ;:,-:-~. When inoculating, all tiroths should ~ethyl Red & Vogues-Proskau_cr receive a single colony from an Methyl red is a pH indicator and 1s used to ? isolation plate. All slants should be detect mixed-acid fermentation by bacteria. streaked and stabbed with bacteria After growth, when it is added to a culture from a single colony. All deeps should with a pH below 4.5, the broth in the tube be stabbed with bacteria from a single will turn bright red. This is the only positive colony. Flame the loop between each result. If the solution is orange it is considered and every inoculation. inconclusive or negative. No color change is considered a negative result, as well. ? After growth, add 3 drops of Methyl Red to the test tube. Swirl gently. The reaction will turn red almost immediately if the mixed acid test is positive. Vogues-Proskauer is a test for the products 2,3-butanediol and acetoin. When Barritt's reagent A and B are added to the tube, the presence of these products will react and turn red. ? After growth, add 18 drops of Barritt's Reagent A and 18 drops of Barritt's reagent B to the test tube. Swish vigorously to get the reagent to oxide the 2,3-butanediol and acetoin, if present. Set a timer for 30 minutes. You can ONLY read the result AFTER 30 minutes has passed. FIGURE 150 26.4 Methyl Red test, positive, negative. FIGURE 26.5 Vogues-Proskauer test, positive, negative. An Inquiry-Based Guide to Experimentation in Microbiology Passaic County Community College 2. Catalasc Catalase is an enzyme that is used by bacteria to break down hydrogen peroxide into gaseous oxygen and water. Using good aseptic technique, take some bacteria from a tube or plate and transfer it to a slide. Add 2 drops of hydrogen peroxide and watch the bacteria to see if it fizzes or bubbles. If catalase is present, bubbling will occur within 3-5 minutes, often immediately. Alternately, 2 drops of hydrogen peroxide can be dropped onto a colony on a plate, slant or deep, but be mindful that the results of such action may ruin the plates or tubes for use in further study. FIGURE 26.6 Catalase test, positive, negative. /J· Nitrate Reduction Some bacteria can use the oxygen found in nitrate as an electron acceptor during anaerobic respiration and this means that the bacteria are facultatively anaerobic. This reduction of nitrate to nitrite is performed by an enzyme called nitratase. In order to test for nitrate reduction, we check for the presence of nitrite in the test tube. Reagent A and 3 drops of Nitrate Reagent B. Nitrate A has sulfanilic acid and Nitrate B has dimethyl-alpha-naphthylamine. These reagents will cause the broth to turn red if there is nitrite produced by the bacteria. '. ~..- .... ' . . :. . - '_ . • . . ? After growth, add 3 drops of Nitrate .,,, .~ .·; . 'I' ~ •. . / r ,. ' ···-~-:: .;, .. FIGURE 26.7 . ~ . •.,_~. :~... .~ ____ ;:_ L ' l ·-. ' . ' ·. -..-.1 Nitrate test, positive, negative. If the tube does not change color, the negative reading must be confirmed. The addition of a little bit of zinc dust or zinc shot will change nitrate to nitrite which will then react with the reagents and turn red within 10 minutes. If the test still does not change color, the ba(..'teria are assumed to have reduced nitrogen to ammonia, nitric oxide, nitrous oxide, or completely to N 2 gas. ? 4."'Sulfur Reduction (SIM) ~ The SIM tube is used as a test for 3 different bacterial characteristics: the sulfur reduction, indole production, and motility. Since it is an agar deep, it is also part of determining the oxygen requirements of the organism. Sulfur reduction will occur if the bacteria have one of two enzymes; cysteine desulfurase or · thiosulfate reductase. Sulfur reduction will create H 2S gas and if iron is present, a black precipitate in the tube. The precipitate might look like generally black agar, or it might look like little flecks or pepper flakes. If the bacteria are capable of breaking down tryptophan, called tryptophan hydrolysis, it will be due to the presence of an enzyme tryptophanase. This enzyme's products are pyruvic acid, ammonia, and indole. The addition of Kovac's Reagent will react with the indole, if present, and appear as a bright red band at the top of the tube. • Add .8 drops of Kovac's Reagent to the top of the tube. The presence of indole in the media will create a red band on the top of the agar. This indicates the presence of the enzyme tryptophanase. Lab 26 Project-Identifying an Unknown Bacterium 151 tic . . . rhe lasr characteris b . Motility 15 from this tu e. ined eterm · . that can be d rhe bactena to swim of bl earance h The a 1 ,ty will be seen by a azy app stab rire length of the . along che en . c.he aga.r If the bactena are non. into .1 they will not be able to swim f mot1 e, into the agar and the appeara~ce che tube will show a sharp kmfe-hke e tube where the loop . cut cut down th the agar. The ability to determine motility in this manner als~ depends on the ability of the bactena to grow throughout the oxygen gradient inside the tube. Obligate aerobes are more difficult to study for motility in this way, since they only grow at the top of the tube, regardless of their ability to "swim." .·~ ·.. ,, ? . FIGURE 26.8 SIM tube·, indole-positive red color occasionally appears in botto m of tube when the agar cracks. Right tube negative for indole with band on top. . FIGURE 26.9 Tube showing non-motile and motil e bacteria. 5. Urea Hydrolysis Urea can be split by an enzyme calJed urease to make ammonia and carbon dioxide. Ammonia in the media will turn the phenol red indicator pink, and therefore represents a positive test for this enzyme. Urea hydrolysis broth and urea hydrolysis agar are not the same, however. The agar can take a week to fully develop in order to be read, but the broth needs to be . read in 24 hours. FIGURE 26.1 0 Urea broth throu ghou t pH range is Furthermore, if the organism not capable of rapidly utilizing its urease users to grow. The broth is a rapid urease, there is not enough food in the tube to allow slow to check a broth 24 hours after the test is test, the agar is more comprehensive. If there is not time the next lab session. For the most started, arrange to move the tubes to the refrigerator until be used. thorough determination of urease presence, agars should 152 · Ant ogy nqu,ry-Based Guide to Experimentation in Micr obiol Passaic Coun ty Comm unity College 6. Kligcr's Iron Agar (KIA) There a rc many different compo nents t h' glucose, Iactose, pcpto ne fro m anima l so t is _ tube's. media ·