Unknown lab report 2J Final copy PDF

Preview

Summary

Lab Report- Example ...

Description

Citrobacter freundii

Severe 1

Unknown Lab Report Unknown Number: 2J Jennifer R. Severe Microbiology & Public health 2310L-021 Georgia State University Perimeter College-Clarkson Campus July 25,2019

Citrobacter freundii

Severe 2

Introduction: “Microbes” are all around “us.” Microbes also known as microorganisms(bacteria) are “minute living things that individually are usually too small to be seen with the unaided eye” according to Gerald Totora in the textbook “Microbiology” 13th ed. (Totora, 2). Living in a world where microorganisms are the prime predators of disease-causing illness and deadly viruses, it is vital that microbiologist, scientist and lab technicians understand the proper safety procedures, tests, experiments such as Koch’s postulates for example to determine the causative agent of an specific pathogen may cause the same diseases in which the specimen was collected from in humans, animals and plants. (Totora,399). In following these procedures, lab experimenters will be able to determine the proper treatment needed to treat the disease, illness or subside the symptoms of a virus being observed that is affecting all forms of life. It is also vital that experimenters such as microbiologist, scientist and lab technicians use mandatory techniques such as aseptic technique; the process of using a sterile inoculating loop or needle to transfer or isolate an culture into a broth, slant or agar plate in order to prevent contamination of an microorganism and to also reduce possible infection to the experimenter and colleagues whom are testing an specific microorganism.(McAllister,6) In experimenting an unknown disease-causing microorganism, one test that experimenters most often use and is usually the first differential test ran on a specimen that is brought into a laboratory is Gram stain (1) according to Micheal J. Leboffe, et.al ( Leboffe-et.al, pp 45-46). This test may be used to determine a microorganism’s cell morphology such as shape ((Spirillum (spiral, curved), Bacillus (rod) and Coccus (round)), size, cell arrangement (single, di, tetrad, cluster, etc.) and is significantly used to distinguish between Gram-positive and Gramnegative cells and their wall structure and type. According to Leboffee is used to identify

Citrobacter freundii

Severe 3

whether an microorganism is an Gram-positive or Gram negative organism, (Leboffe,45) both Gram-positive and Gram-negative microorganism are stained by an primary staining agent called Crystal violet and enhanced by an mordant iodine to form an crystal violet-iodine complex, following with an decolorization agent of either alcohol or acetone to remove the dye crystal violet form Gram-negative cells,(only Gram-negative cells are decolorized from this agent, Gram-positive cells remain unaffected and are not decolorized by alcohol or acetone (Leboffe,45). The counterstain of safranin is then used to dye Gram-negative cells. (Leboffe,45) After successful completion of the Gram-staining tests, experimenters will be able to identify whether a specimen is either Gram-Positive by a purple coloration indicator under microscope or Gram-negative by red or pink coloration under microscope. Experimenters can also distinguish between a Gram-positive and Gram-negative specimen by observing the structure of the cell walls of the stain.(Leboffe,45) As stated by Leboffee, under careful observation under a microscope and other evidence to indicate that the ability of a specimen to resist decolorization or not is based on the “different wall structures of Gram-positive and Gramnegative cell walls”. (Leboffe,45). “Gram-negative cells walls have an high amount of lipid content and thinner peptidoglycan layer than Gram-positive cell walls due to having an outermembrane” in which Gram-positive cells does not have, and the “decolorizer alcohol/acetone extracting its lipid content making the Gram-negative wall more porous and in capable of retaining the crystal violet– iodine complex, therefore decolorizing it” (Leboffe,45). Grampositive cells have a “thicker peptidoglycan layer than Gram-negative cells and due to this it is less susceptible of decolorization because of the thick peptidoglycan layer cross-linking due to teichoic acids trapping the crystal violet -iodine complex”. (Leboffe, 46).

Citrobacter freundii

Severe 4

Though gram stain can determine the gram reaction of an specimen due to coloration and morphology of the specimen being observed, it sometimes may not be reliable to properly identify an unknown specimen due to an experimenter improperly following the gram stain procedures and causing a gram-negative stain to look gram positive, if crystal violet was overused, or the decolorization and counterstain agent was not used.(Leboffe,46) Moreover, though the gram stain test (1) is usually the first and most highly used tests in an lab to identify an unknown specimen, (Leboffe,45) experimenters also use other tests such as the fermentation tests (2), which is used to determine whether or not bacteria can ferment a specific carbohydrate (tankeshwar,2016). The fermentation tests according to Totora uses a test medium that contains protein, a single carbohydrate, a pH indicator, and an inverted Durham tube, which is used to capture gas and identify the presence of gas in a specific carbohydrate (glucose, lactose and sucrose) being tested. (Totora,134). Not only does the fermentation test; test for the presence of gas, it also tests for the presence of acid production due to the various pH indicators that can detect acid production from fermentation. (Totora,134). If the test changes color after incubation, by use of an “phenol red broth or purple broth, and a yellow change of color is observed then this indicates acid production by a carbohydrate, which lowers the pH level to 6.8, turning the medium yellow (Leboff,71) (Mcallister,20). The presence of a pink coloration changes or no color change (RED), indicates the carbohydrate is alkaline and is above the pH indication level of 7.4 due to the deamination of the peptone amino acids producing ammonia to raise the pH indication level in the fermentation test. (Leboff,71) (Mcallister,20). In, all the fermentation test is primarily used to differentiate members of the Enterobacter family and distinguish from other Gram-negative rods; that can be done by examining the different patterns of acid, gas, or coloration that

Citrobacter freundii

Severe 5

occurred during the testing period. Glucose, sucrose and lactose are the most commonly used sugars and they accomplish fermentation by glycolysis to present an end product of acids, alcohol, and H2 or CO2 gases. (Leboff,71). Though, the fermentation test is reliable in giving experimenters the best possible results of what an unknown specimen may be by being able to differentiate each microorganism from each other due to given results by fermentation, it is not re liable for accurate confirmation of fermentation if the test is read after 18-24 hours (Leboffe,71). The Citrate utilization test (3) is used to determine the ability of an organism to use citrate as its sole source of carbon and distinguishes between members of the family Enterobacteriaceae and differentiates them from other Gram-negative rods. (Leboffe,64). The presence of citrate or citric acid in many bacteria is produced by “acetyl coenzyme A” from oxidation fatty acids which reacts with oxaloacetate at the entry of the Krebs cycle,” (Leboffe,64). The Citrate utilization test uses a medium that contains only citrate as the available source of carbon for a specimen being tested. (Leboffe,64) The Citrate test uses a pH indicator called Bromothymol blue dye, which is “green at pH 6.9 and blue at pH 7.6” to indicate bacteria that “possess citrate-permease; the ability of an bacteria to transport molecules into a cell and enzymatically convert it into pyruvate”. (Leboffe,64). The end products of the pyruvate can then be examined in numerous ways depending on the pH of the environment. The Citrate utilization test uses, a specific agar called “Simmons Citrate Agar” an defined medium that contains sodium citrate as the sole carbon source and ammonium phosphate as the sole nitrogen source. (Leboffe,64) (Mcallister,24). Bacteria that survives on the medium and utilize citrate changes as stated above changes color from green to blue by way of pH level in the environment. The conversion of ammonium phosphate to ammonia (NH3) and ammonium hydroxide (NH4OH) on

Citrobacter freundii

Severe 6

the citrate medium alkalinize the agar, the coloration of green (negative reaction) on the agar (remaining the same) indicates that bacteria will not survive in this environment and does not utilize citrate due to low pH levels, while the presence of a blue (positive reaction) coloration on the agar indicates that bacteria can survive on the medium and utilizes citrate due to the high pH levels of 7.6 or more (Leboffe,64). The Citrate utilization test is a reliable test to distinguish some Enterobacter, but some bacteria may still utilize citrate and not produce a color change according to Michael J. Leboffe (Leboffe,64). The Indole test (4) is used to identify bacteria capable of “producing indole by using an enzyme called tryptophanase and differentiate Enterobacteriaceae family members” (Leboffe,74). The indole test is a component of the IMViC battery test, and production of indole on a medium is indicated by the presence of tryptophan (animal protein). (Leboffe,74).The hydrolysis of tryptophan in the broth can be detected according to Leboffe by adding an reagent called Kovac’s reagent to the medium, Kovacs’ reagent contains “dimethylamine benzaldehyde (DMABA), which when a few drops of Kovac’s regent is added into broth, it forms a liquid layer over the solid medium, DMNBA which reacts with the presence of indole and produces a quinoidal compound that turns the reagent surrounding layer (red)”.(Leboffe,74)(Mcallister,24). The presence of a red color in the indole test’s reagent layer indicates a positive reaction and the presence of tryptophanase, the presence of yellow or no color presence indicates a negative indole-reaction. (Leboffe,74) (mcallister,24). The indole test can also be used “alternatively” according to Leboffe by “placing bacterial growth on a paper slide impregnated with 5% DMABA, a positive result is indicated by a formation of pink on the paper slide” (Leboffe,74). The indole test is one of the most reliable in the IMViC battery test.

Citrobacter freundii

Severe 7

Voges Proskauer a.k.a VP (5) is used to identify “organisms that are able to produce acetoin from the degradation of glucose during a 2,3-butanediol fermentation” (Leboffe,98) and “used to detect two different types of glucose fermentation” (Mcallister,24). Like indole, VP is also a component of the IMViC battery test which helps experimenters distinguish between members of the Enterobacteriaceae family and differentiate them from other gram-negative rods (Leboffe,98). VP (5) as stated in the atlas is a simple solution containing only peptone, glucose, and a phosphate buffer. “The peptone and glucose provide protein and a fermentable carbohydrate, and the potassium phosphate resists pH changes in the medium or broth” (Leboffe,98). The addition of VP reagents to a broth medium containing the specimen being examined after the incubation period oxidizes acetoin only occurs if present to diacetyl, which reacts with guanidine nuclei from peptone to produce a red color, indicating a positive VP (5) result. (Leboffe,98). A negative VP result either has no color change or forms a cooper-like or light pinkish coloration which results from the interaction between the reagents used, the “pinkish color indication of a negative result may be confused for a positive reaction” (Leboffe,98), if the experimenter does not interoperate the results or data correctly. In all the VP test is a reliable to detect which Enterobacter organisms are “able to ferment glucose and quickly convert their acid production into acetoin and 2,3-butanediol”(Leboffe,98) in order to find the correct bacteria that is being observed as an unknown specimen but can be confused if an negative result shows a light pink color change. Moreover, these (5) test described above were used and tested on an unknown specimen in a microbiology lab to identify specimen from its enterobacteria family and group. These tests are reliable in identifying and distinguishing difference between microorganisms in a similar

Citrobacter freundii

Severe 8

family but sometimes can be unreliable if the experimenter misses a step in the procedures, contaminate the medium or the organism doesn’t show the proper color indication.

Objective: In performing these experiments, an unknown bacterium (specimen) can be identified and determined.

Hypothesis: (1). If the bacterium in the tube (2J) stains pink or a mixture of pink and purple and it’s cell morphology is a rod shape then the bacterium is a Gram-negative microorganism and (8) microorganisms can be the possible unknown specimen in the tube (2J):Enterobacter aerogenes (1), Escherichia coli (2), Serratia marcescens (3), Citrobacter freundii (4), Proteus vulgaris(5), Proteus mirabillis (6), Pseudomonas aeruginosa (7) and Pseudomonas fluorescenes (8). (2). If the bacterium in the tube (2J) after testing for alkaline, gas and acid production in a phenol red broth fermentation test: glucose, lactose and sucrose has gas and acid production for glucose and sucrose (AG) and acid production for lactose(A), then (4) microorganisms out of the (8) possible can be the unknown specimen in tube (2J); these possible microorganism are Serratia marcescens (1), Citrobacter freundii (2), Proteus vulgaris(3), Proteus mirabillis (4). (3). If the bacterium in tube (2J) is Serratia marcescens (1) after adding Kovac’s reagent to a broth inoculated with the specimen in the indole test has a red surface layer, then it will have a positive result reaction to future tests, Citrate Utilization and VP. If the bacterium is Citrobacter

Citrobacter freundii

Severe 9

freundii (2) and a yellow surface layer is observed during the Indole test, then it will have positive result for citrate utilization and negative result for VP. If the bacterium is Proteus vulgaris (3), and red surface layer during the Indole test is observed then it will have a negative result for the Citrate utilization test and VP test. If the bacterium is Proteus mirabillis (4) and a yellow surface layer is observed during the indole test, then it will have an unknown reaction for the Citrate utilization test and a negative reaction for the VP test. (4). If the bacterium in the tube (2J) during the Citrate utilization is Serratia marcescens then it will turn blue after inoculating and incubating the specimen for more than 24 hours indicating a (positive reaction). If the bacterium is Citrobacter freundii (2), then it will turn blue, indicating a (positive reaction). A positive reaction in citrate utilization test indicates the microorganism utilizes citrate. If the bacterium is Proteus vulgaris (3), then it will have a negative reaction, and therefore will be eliminated if after testing the results are blue. If the bacterium is Proteus mirabillis (4) as a possible specimen in tube (2J) then it will also be eliminated from the list of possible organisms; since its reaction is unknown. If after testing the unknown specimen and the results are blue, then (2) possible microorganism may be the specimen in tube (2J); Serratia marcescens (1) and Citrobacter freundii (2). If after testing the unknown specimen, and the results has no color change and remains green, then the possible organisms in tube would be narrowed to Proteus vulgaris (3) and Proteus mirabillis (4). (5) If the bacterium in the tube (2J) after testing for a positive or negative reaction in the Voges Proskauer test turns red indicating a positive carbohydrate fermentation, then the organism is Serratia marcescens (1), due to having a negative result for Indole test and positive result for Citrate utilization as listed in the identification key chart. If the VP test shows a negative result, then Serratia marcescens will be eliminated as the possible specimen in tube (2J). If the

Citrobacter freundii

Severe 10

bacterium is negative in the Voges Proskauer test, shows no color change or has a copper-pinkish color indication, showing that the organism does not ferment carbohydrates, then organism is Citrobacter freundii, due to having a negative indole test reaction and positive Citrate Utilization test result. If the VP test shows a positive reaction, then Citrobacter freundii will be eliminated as the possible specimen in the tube. If the bacterium in the tube (2J) is Proteus vulgaris (3) then it will have a negative VP test result, which will indefinitely eliminate it as a possible bacterium in tube (2J) since it had positive indole and negative Citrate utilization test result. If the bacterium in the tube is Proteus mirabillis (4), then it will have a negative VP test result, and may possibly be eliminated from the list of possible organisms to be the bacterium in the tube 2J since it results for the Citrate utilization test is unknown and the carbohydrate fermentation for sucrose is also unknown. (6.) If after all testing results is complete and the results are: negative for indole, positive for VP, and positive for Citrate utilization, then the bacterium in tube (2J) is Serratia marcescens. If the results are: negative for indole, negative for VP, and positive for Citrate utilization, then the bacterium is Citrobacter freundii. If the results are: negative for indole, negative for VP, and unknown for Citrate utilization then bacterium cannot be a possible organism in the tube 2J, since some of its results are undefined or unknown. If the results are: positive for indole, negative for VP, and negative for Citrate utilization, then Proteus vulgararis, cannot be a possible organism in the tube (2J) since the results observed and gathered are opposite of its indole and Citrate utilization test and it was eliminated after the Citrate utilization test results.

Citrobacter freundii

Severe 11

Procedure: Step 1: In the process of identifying an unknown bacterium chosen as witnessed by the lab instructor Dr. Falkenberg, a reserve stock of the unknown specimen working stock tube (2J) was made and labeled 2J(R) and then incubated at 37℃ for 48 hours and until the next lab session. Step 2: In testing the possible unknown bacterium in the tube (2J), gram stain was tested twice to identify and confirm the accurate gram-stain reaction and morphology of the organism. In the gram stain test, “the primary reagent (crystal violet), enhancer (iodine), decolorizer (alcohol/acetone) and counterstain (safranin)” were used to identify the stain reaction and morphology for the unknown specimen (2J) in order. (Mcallister,14) Step 3: The fermentation was used to identify the unknown specimen in tube (2J) and so the specimen was inoculated from the tube (2J) into a phenol red broth containing glucose, lactose and sucrose to “test for possible alkaline, gas or acid production”. (Mcallister,20). Step 4: Repeat: The gram-stain was repeated after the fermentation test resulted in possible organisms for the unknown specimen (2J) being gram-negative and should not stain purple, the process of gram-staining was repeated until proper amounts of the reagents were added in a more proper timely manner to give the accurate result of a gram-negative stain. Step 5: In the Indole test, the unknown specimen in tube (2J) was inoculated into a “tryptone broth and incubated until the next lab session”, after “1ml” of the was taken from the broth and added into a sterile empty tube, following with “4 drops of Kovac’s reagent”. (Mcallister,24)

Citrobacter freundii

Severe 12

Step 6: The Citrate utilization test was used to also identi...