This action might not be possible to undo. Are you sure you want to continue?
Praise Selah G. Dagoc Mambajao, Camiguin
Identification of microorganisms from unknown sample is a routine work for a Registered Medical Technologist assigned in the Microbiology section of the laboratory. For medical technology students, this activity serves as an important tool in exercising the students skills and knowledge in the processing and analysis of the various methods used in proper identification of isolates. In this activity, the student was able to identify Proteus vulgaris from an unknown sample with the use of scientific steps and procedures for proper identification of microorganisms in bacteriology.
Through this activity, the student should be able to: y Practice skills on proper identification of microorganisms from sample. y Properly execute the steps in identification of microorganisms. y Know the background and diseases associated with the microorganism isolated and identified from the sample. y Describe the characteristics of the isolate. y Discuss the infections caused by the isolate and its treatment.
environmental habitats, including long-term care facilities and hospitals. In hospital settings, it is not unusual for gram-negative bacilli to colonize both the skin and oral mucosa of both patients and hospital personnel wherein infection primarily occurs. (Struble, et al., 2009) Patients with recurrent infections, those with structural abnormalities of the urinary tract, those who have had urethral instrumentation, and those whose infections were acquired in the hospital have also been found to have an increased frequency of infection caused by Proteus and other organisms like Klebsiella, Enterobacter, Pseudomonas, Enterococci, Staphylococci. (Struble, et al., 2009) P. vulgaris is an enteric bacterium, which means it is found in the intestinal tract (Deacon, J.); it is also found in the soil, contaminated water, or decomposing organic substances (University of Texas, 1995). While it is a pathogenic bacterium and has been shown to cause urinary tract infections, it is also part of the natural flora of the intestinal tract (Struble, et al., 2009); and because the microbe can live on the skin of some people, P. vulgaris is a common pathogen in hospital wound infections, especially in the immunosuppressed. (Struble, et al., 2009) Conversely, Proteus vulgaris is easily isolated from individuals in long-term care facilities and hospitals and from patients with underlying diseases
Proteus vulgaris is one of the most commonly isolated members of Proteus sp., along with Proteus mirabilis. The genus Proteus is a member of a large gram-negative bacilli family, Enterobacteriaceae. Proteus organisms are known to be one of those to cause serious infections in humans, along with Escherichia, Klebsiella, Enterobacter, and Serratia species. (Struble, et al., 2009) Proteus species are normal flora of the human intestinal tract, along with Escherichia coli and Klebsiella species, of which E. coli is the predominant resident. Proteus is also found in multiple
Page | 1
it has an extracytoplasmic outer membrane. it is not susceptible to ampicillins or cephalosporans. because it is a facultative anaerobe. several tests can be used. J. vulgaris is a chemoheterotroph. et al. Unfortunately. it is not the most likely candidate for community-bourn disease. The bacterium grows and stops in waves. (Struble. It creates an endotoxin. It will test positive on the citrate test (Deacon.Triple Sugar Iron (TSI) Agar . the glucose fermentation only occurs in anaerobic conditions. (Struble. vulgaris. 2009) The bacterium is a gram-negative rod with flagella. Methyl Red Voges-Proskauer Medium. 1995) The reason the urinary tract is such a hospitable environment for the colonization of the microbe includes the microbes ability to degrade urea to ammonia with the enzyme urease. while P. 2009). the following materials were used: y Sheep Blood agar plate (SBAP) y MacConkey Agar plate (MAC) y Test tube containing a Tryptic Soy broth with an unknown sample y Test Battery for identification of gram negative bacilli: . et al. the microbe will use a variety of organic molecules to survive. creating what appears to be distinctive rings in the manner of tree rings. et al. Struble. (Deacon. 2009). Likewise. J. As a gram-negative rod. 2004) These biochemical properties and physical observations helped in the indentification of the unknown. (Deacon. it ferments glucose but not lactose or mannitol.. which turned out to be P.Lysine Iron Agar (LIA) .) When identifying the microbe. as a chemoheterotroph. 2004).) and urease test (Struble. (American Academy of Family Physicians. it will be noticed on non-selective media a "swarming" behavior. which allow it to be extremely motile. Because it ferments glucose but not mannitol or lactose.. 2009) P. each bacteriology student was given a Tryptic Soy Broth containing an unknown Page | 2 . and Simmons Citrate Agar) . This feature is the result of the microbe s flagella. which means it uses carbon sources like glucose for energy and carbon (American Academy of Family Physicians.Urea broth . 2009) But. when observing the plated colonies..). it will only test positive in the glucose/carbohydrate utilization tests..OF-Carbohydrates (Mannitol. Sucrose. (University of Texas. (Struble. where the microbe grows in waves (Deacon. which can cause a deadly systemic inflammatory response in 20 to 50 per cent of its victims. 2004) However.Phenylalanine Deaminase Agar Slant .Identification of Proteus vulgaris from Unknown Sample or compromised immune systems. if placed in non-ideal. However. et al... (Clayton State University. Lactose???) y Inoculating loop y Inoculating needle y Alcohol lamp y Test tube rack y Forceps y Glass slides Methods As a part of the final activity for the bacteriology laboratory class. vulgaris does contribute to the Proteus infections. aerobic conditions.IMViC (Sulfide Indole Motility medium. et al. P. vulgaris is the lead pathogen in causing hospital-bourn Proteus diseases. 2009) It has been shown that its optimal growth temperature was at 37 C. Materials To identify the bacteria from the unknown sample. J.. Proteus mirabilis causes a majority (90%) of those diseases (Struble. et al. J. The plate will also smell like burnt chocolate.
circular. giving the student a guide on what to expect. gram stain was applied following the appropriate order and time for each reagent: Crystal Violet (primary stain) 1 minute. Colonies observed were pink rods. On phenylalanine slant. Whereas. gram staining was performed to identify gram stain reaction of the isolate and guide in biochemical testing. 1-2 drops of methyl red were put into the MR tube. Amino Acid Degradation tests (Phenylalanine & Lysine Iron Agar). and were arranged in singles. After inoculation.Identification of Proteus vulgaris from Unknown Sample isolate which the student must identify using the appropriate methods he/she has learned from class. 4-5 drops of 10% aqueous ferric chloride was added.. the test batteries were removed from the incubator and results observed. Urea hydrolysis test. On the following day. the student worker performed biochemical testing using the test battery for gram negative bacilli which consists of: IMViC (SIM. The author received the TSB tube number 8. Gram negative (pink) rods (figure 2. the slide was examined under the microscope set on oil immersion objective. The loop was used to get a part of the colony and aseptically transferred into each broth by tapping the loop inside the tubes. undulate pink colonies were observed in the MacConkey Agar plate (figure 1. Results Swarming. On SIM. flat. A colony from SBAP was transferred onto a pre-heated (sterile) glass slide with the use of a sterile inoculating loop. This activity serves as an application of all the lessons that were taught and to test if the student understood and remembered the various tests that were mentioned in the laboratory and lecture classes in bacteriology.3) whereas swarming. Full stabs were done on the OF tests.2). transfer was done using a sterile inoculating needle by stabbing the butt area and streaking the slant in a zigzag pattern. 95% Ethyl Alcohol (decolorizer) 30 seconds. indicating gram negative reaction. Because of this finding. These tubes were then placed in an incubator and incubated at 37rC for 24 hours. This colony characteristic illustrates a presumptive identification of a Proteus sp. Triple Sugar Iron Agar. MAC was directly placed into the incubator and incubated at 37rC for 24 hours.2) were seen on the gram stain of a colony from SBAP.1 & figure 2. sterile inoculating needles were used to transfer samples from the plate (MAC) into the solid and semi-solid media. Inoculation on citrate and phenylalanine was done by streaking the slant in a zigzag pattern using a sterile inoculating needle. Gram s Iodine (mordant) 1 minute. and 10 drops of 5% alpha-naphthol followed by 15-20 drops of 40% KOH were added to the VP tube. gray colonies were seen on the Sheep Blood Agar plate (figure 1. Upon drying of the stained smear. SBAP was placed in a candle jar and incubated at 37rC for 24 hours. For MR-VP. On the first day of the activity. Page | 3 . and Carbohydrate Fermentation Tests (Mannitol. etc. Stabbing was done twice in LIA after which the slant is streaked in a zigzag pattern only once. MR-VP & Citrate). Next. and Safranin (counter stain) 45 seconds. These colonies were found to be arranged in singles and clusters. Smearing was done by spreading the colony in a circular motion.1 & figure 1.????). the student inoculated sample using an inoculating loop from the TSB broth into the Sheep Blood Agar Plate (SBAP) and MacConkey plate (MAC) through the multiple interrupted streak method. 2-5 drops of paradimethylaminobenzaldehyde (PDAB) were added for Indole test. The smear was then air-dried and heat-fixed before staining. flat. The next day. SBAP and MAC were removed from the incubator and colony appearance from each of the plates was noted. Afterward. Transfer into the SIM medium was done by stabbing the medium until 3/4th to the bottom. Sample was inoculated from MAC plate into MR-VP and Urea using a sterile inoculating loop. pairs and clusters. On the other hand. On TSI.
(Struble. Complicated UTIs in men and women can be treated with a 10. SIM showed a nonmotile reaction demonstrated by the lack of a brushlike appearance. For phenylalanine deaminase test.. ferm.4) as shown in the development of a redviolet color in the urea tube. Fermentation (yellow color) was seen on OF-Sucrose and OF-Maltose whereas OFMannitol showed an inert reaction evident in the presence of a blue color near the surface of the butt. and airway Page | 4 . cephalosporin. therapy consists of parenteral (or oral once the oral route is available) ceftriaxone. the recommended empirical treatment includes an oral quinolone for 3 days or trimethoprim/sulfamethoxazole (TMP/SMZ) for 3 days for uncomplicated UTIs in women on an outpatient basis. Sulfide reaction was also observed as demonstrated by the blackening of the medium (See figure 3. anaphylactoid) reactions have occurred in patients receiving antibiotics.5) as demonstrated in the blackening of the medium (A/A H2S).. A positive reaction (red color) was seen in the MR tube whereas VP tube exhibited a negative reaction (no change in color). Oxygen. These reactions are more likely to occur in persons with a history of sensitivity to multiple allergens. et al. Crosssensitivity between penicillins and cephalosporins has occurred. With the addition of 2-5 drops of paradimethylaminobenzaldehyde (PDAB). Gas production was not seen in TSI. H2S reaction was seen in TSI butt/slant (figure 3. discontinue the implicated drug unless the condition is life threatening and amenable only to therapy with that antibiotic. or TMP/SMZ for 14 days may be added to complete treatment. Biochemical Test Results Sulfide + SIM Indole + Motility MR + VP Cit + Urea + LIA . Ultrasonography of the kidneys or a CT scan could also be considered as part of a workup for Proteus infection of the urinary tract that does not resolve quickly with antimicrobial therapy.3) showed a positive reaction as evident in the prussian blue color of the agar slant. 2009) However. a K/A reaction or negative lysine decarboxylation was observed (purple slant/yellow butt). If a reaction occurs. red color developed on the surface indicating a positive reaction for Indole. quinolone. Calices and/or perinephric abscesses should be excluded. For hospitalized patients.Identification of Proteus vulgaris from Unknown Sample Table 1-1. Then. intravenous steroids. or aztreonam until defervescence. Discussion For the biochemical testing. 2009) For treatment. an oral quinolone. (Struble. et al.1 in appendix). The isolate was also found to be positive for urea hydrolysis (figure 3. For MR-VP (figure 3.2). 4-5 drops of 10% aqueous ferric chloride was added to the slant after which a green color developed indicating a positive result.6). Citrate tube (figure 3. A summary of the biochemical test results is shown in table 1. ferm. serious and occasionally fatal hypersensitivity (ie. or an oral cephalosporin or quinolone for 14 days as outpatient therapy.(K/A) Phenyl + TSI + (A/A with H2S) OF-Carbohydrates SUC MAL MAN ferm. single-dose ceftriaxone or gentamicin followed by TMP/SMZ. gentamicin (plus ampicillin). In addition to these methods.to 21-day course of oral therapy (in the same manner as for hospitalized patients) as long as the follow-up is adequate. Acute uncomplicated pyelonephritis in women can be treated with oral quinolones for 7-14 days. Serious anaphylactoid reactions require immediate emergency treatment with epinephrine. In the LIA butt/slant (figure 3. 1-2 drops of methyl red were put into the MR tube and 10 drops of 5% alpha-naphthol followed by 15-20 drops of 40% KOH were added to the VP tube.
et al.ac. (2009).Identification of Proteus vulgaris from Unknown Sample management... uropathogens are resistant to the drugs used in these systems.com/med/ byname/proteus-infections. aminoglycosides. Broad-spectrum antimicrobial therapy is started empirically and is modified by the results of smears and cultures.med. including intubation. Houston (1995). "Proteus Infections..com. even with adequate treatment. as other. et al. K. the student concludes that the organism isolated was Proteus vulgaris. and quinolones have excellent activity (90%-100%). The Microbial World: Proteus vulgaris and clinical diagnostics. 2009) Most nonurologic infections result in abscesses. References Struble. Retrieved on March 16. typically a urologist or nephrologist. Bacterial Identification. Mortality and morbidity rates are high.edu/path/00001517. et al. Retrieved on March 16. TMP/SMZ.uk/bto/microbes/ roteus.emedicine.and longterm management of stones." Emedicine. Retrieved on March 14. et al. infections caused by P.4 While the use of these types of catheters for Proteus UTIs is helpful in containing the migration of Proteus in experimental models. an organism commonly associated with urinary tract infections. et al. et al. Imipenem.xml.. American Academy of Family Physicians 2004. et al. Jim.bto. surgery. fourth-generation cephalosporins. 2010 from http://www. 2009) In addition.htm. 2009) P. 2009) Summary and Conclusion Based on the different tests performed. is resistant to ampicillin and first-generation cephalosporins. 2009) In presence of struvite renal calculus associated with Proteus infection. vulgaris. htm. University of Edinburgh. penneri. Moreover.ed. 2010 from http://helios. vulgaris are treatable with antimicrobial drugs and.tmc..3. Retrieved on March 16. in the case of renal stones. Institute of Cell and Molecular Biology. Activation of an inducible chromosomal betalactamase occurs in up to 30% of these strains. AAFP. Deacon. but it is not available commercially. University of Texas. (Struble. (Struble. (Struble. but tissue recovery is often better than expected. 2010 from http://www. (Struble. 2010 from http://medic.com. (Struble..aafp.uth. et al. 2009) A vaccine derived from purified mannoseresistant Proteus -like (MR/P) fimbriae proteins has been proven to prevent infection in mouse models and is under clinical research. Radical surgical debridement is the cornerstone of successful therapy. 2009) The discovery of stones requires an evaluation by a physician knowledgeable in the short. should also be used as indicated..org/x2215. along with P. (Struble. Proteus. Page | 5 . (Struble.. this practice is not widespread. surgery must be done to remove it. the use of chlorhexidine and triclosan in closed urinary catheterization systems and drug-impregnated catheters reduce the incidence of Proteus UTI in patients with long-term indwelling urinary catheters. Amputation may be necessary if skin or muscle necrosis of an extremity is the presenting infection. more common.htm.
Culture Plates Figure 1.3 P. undulate characteristic of the P. Colonies show a swarming. Page | 6 . gray-colored appearance Figure 1. Figure 1.Identification of Proteus vulgaris from Unknown Sample Appendices Appendix 1. vulgaris colonies.1 P.vulgaris colonies on Sheep Blood Agar plate.vulgaris colonies on MacConkey Agar show the characteristic pink swarming colonies.2 A closer look on the colonies in SBAP will show the flat.
Page | 7 .Identification of Proteus vulgaris from Unknown Sample Appendix 2 Gram Staining Figure 2. pairs and clusters. arranged in singles. Figure 2.2 Gram negative rods of Proteus vulgaris.1 Gram staining of the isolate shows gram negative (pink) rods.
Indole (reddish/pinkish surface upon addition of Kovac s reagent). vulgaris shows a positive reaction for H2S.5 TSI Triple Sugar Iron tubes with P. evident in the blackening of the slant. Figure 3. vulgaris exhibits a positive reaction on Simmon s Citrate medium as shown in the prussian blue color of the agar. vulgaris shows a positive reaction for Sulfide (darkening of the medium). Figure 3. The reaction on TSI is A/A with H2S.Identification of Proteus vulgaris from Unknown Sample Appendix 3 Biochemical Testing Figure 3. vulgaris shows a negative (K/A) reaction on Lysine Iron Agar as demonstrated by the purple slant and yellow butt.6 LIA P.4 Urea P. Figure 3. vulgaris shows a positive reaction on urea hydrolysis test as demonstrated by the red-violet color of the broth. Figure 3. Page | 8 . along with the yellow color on the butt and part of the slant.2 MR-VP Medium MR (left) shows a positive reaction red color upon addition of methyl red. and nonmotile (lack of brushlike growth).1 SIM Medium Isolate of P.3 Citrate P. VP (right) showed a negative reaction no change in color after adding 5% alpha naphthol and 40% KOH Figure 3.
evident in the presence of a green color on the slant after the addition of 10% aqueous ferric chloride. vulgaris isolate as shown in the blue color near the surface of the tube. Figure 3. Page | 9 . Whereas.8 OF-Carbohydrate Tests Fermentation is observed in OF-Sucrose (left) and OF-Maltose (center) as seen in the yellow color of these two tubes.Identification of Proteus vulgaris from Unknown Sample Figure 3. vulgaris is positive on phenylalanine slant.7 Phenylalanine P. reaction in OF-Mannitol (right) is inert in P.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue listening from where you left off, or restart the preview.