How to War Enterococcus Faecalis, Citrobacter Freundii, Klebsiella Pneumonia, and Streptococcus Pyogenes - Lab Report Example

Microbiology Unknowns Lab Report Instructions Manipulative skills and cognitive microbiological knowledge are essential for the identification of microorganism beyond their genus classification, i.e. to their species identification. The use of previously performed experiments and procedures laid the foundation of dichotomous keys, Bergey’s Manual of Systematic Bacteriology. This key aids in the classification of microorganisms and to identify the causal organisms. Further, biochemical tests successfully accomplish the experimental purpose. Species identification can be accomplished by using various established laboratory procedures. Spurious results may be obtained in some cases that departs from the expected norms for a particular species, may be attributed to strain differences within the given species. The unknown samples are required to be handled with care and precision. Identification procedures adopted, identified four bacterial species in the unknown samples, they are: Enterococcus faecalis, Citrobacter freundii, Klebsiella pneumoniae and Streptococcus pyogenes Introduction Microorganisms are ubiquitous and they provide benefit or show their harmful impact on the human/ animal or plant population in varied ways. The body of knowledge that has accrued since these early years has been instrumental in making clinical microbiology a major component of laboratory or diagnostic medicine. It is essential to have an understanding towards these microorganisms for this isolation and identification of these infectious pathogens is imperative. This understanding aids in rapid diagnosis and treatment of the disease, prudently, intelligently and rapidly. Many experiments are being carried out that have relevance with their application in the clinical microbiology. These experiments encompass, isolation and identification of unknown cultures, the use of selective and differential media and various biochemical tests used to separate and identify various microorganisms (Messeley, 2003). The first step to onset the experiment is to perform Gram staining procedure. This segregates the gram positive and gram-negative organisms. This is followed by the streak-plating to observe the colony characteristics. The next step is the use of selective media for the preparation of pure cultures followed by the performance of biochemical tests. The present study was performed to apply all the methodologies learnt in the microbiology laboratory class designed for the detection of an unknown bacterium (Messeley, 2003). Materials Cultures: samples collected in Nutrient broth. This may contain gram-positive or gram-negative organisms. Media: Nutrient agar plates, Nutrient agar slants, Nutrient agar broth, phenyl-ethyl alcohol agar plate and MacConkey agar plate. Reagents: Crystal violet, Gram’s Iodine, 95% ethylalcohol, safranin and other reagents for interpretation of biochemical reactions. Equipments: Bunsen burner, inoculating loop and needle, staining tray, immersion oil, lens paper, bibulous paper, microscope and glassware marking pencil. Methodology Session 1: Separation of the Bacteria in Mixed Unknown Culture Prepare Nutrient agar broth subculture of unknown sample and refrigerate the same. Prepare a gram-stained smear of the original unknown culture. Prepare 4-waystreak inoculations on the following media for the separation of microorganism from the mixed cultures: Nutrient agar for observation of colonial characteristics, Phenyl ethyl agar for the isolation of gram-positive bacteria, MacConkey agar for isolation of gram-negative bacteria. After performing streaking, incubation of these plates in inverted position is performed for 24- 48 hrs at 37° C. Session 2: Preparation of Pure Cultures Colonies obtained on phenyl ethyl alcohol agar plate and on the MacConkey agar plate are aseptically transferred to Nutrient agar slant. This is followed by incubation for 24-48 hrs at 37° C. Session 3: Identification of Unknown Bacterial Species Prepare a gram-stained smear from each of the Nutrient agar slant cultures to verify their purity by means of the Gram reaction and cellular morphology. Incase if the purity is not obtained then session 1 and 2 are to be repeated using refrigerated Nutrient agar culture subcultures using it as test culture. If the isolates are reckoned to be pure on the basis of their cultural and cellular morphologies, continue with the identification procedure. The following dichotomous keys are used for the identification of the organisms (Messeley, 2003). Media Used 1. MacConkey Agar 2. Eosin Methylene Blue agar 3. TSA plate 4. 2 Nutrient Agars (one put in 25C, one in 35 C) 5. Hekton Enteric agar 6. Phenylethanol agar 7. TSI slant 8. M-Ent Agar 9. Simmons Citrate 10. Methyl Red (MRVP broth) 11. Urease Test 12. Catalase Test 13. Oxidase Test 14. SIM media (Sulfur, Indole, Motility) Results Organisms Identified in unknowns mixture: 1. Enterococcus faecalis 2. Citrobacter freundii 3. Klebsiella pneumoniae 4. Streptococcus pyogenes Gram Stains: 1. Enterococcus faecalis: Gram + Cocci 2. Citrobacter freundii: Gram - bacillus 3. Klebsiella pneumoniae: Gram - bacillus 4. Streptococcus pyogenes: Gram + Cocci Media Results MacConkey agar Pink/no fermentation Eosin Methylene Blue agar Light purple & dark purple growth/no fermentation TSA plate Beige growth Nutrient agar at 25C growth Nutrient agar at 35C growth Hekton Enteric agar Yellowish-Orange/no hydrogen sulfide production/ + fermentation Phenylethanol Agar Beige growth TSI slant Yellow/fermentation/hydrogen sulfide production/gas production M-Ent Agar Brown growth Simmons Citrate + blue Methyl Red (MRVP broth) + red Urease Test + Pink Catalase Test negative Oxidase Test Negative SIM Media + sulfur production/No Indole production/+ motility Discussion The main motive to perform this study was to determine the presence of unknown microorganisms present in the provided sample. This study encompasses classification of bacteria based on their chemical reactions temperature, pH levels and osmoregulation. It is very imperative to classify the bacteria. It is also imperative to identify these microorganisms as they have enough potential to cause various diseases. This is essential to carry out the correct diagnosis and treatment of the individual. Identification of the bacteria provides line of treatment. Four bacteria identified in the unknown sample were Enterococcus faecalis is a Cocci and involved in gastrointestinal infections. It is capable of causing infection in community and also in hospital settings. MLST scheme could be used for further confirmation. Moreover phage-typing can also be used for the correct identification of the strain. Moreover the entire genome sequencing can be performed. It is a non-motile organism, it does not show Catalase reaction and display γ-hemolysis, it also display facultative anaerobic nature (Amyes, 2007). Streptococcus pyogenes is also a cocci which shows gram-positive reaction. They are capable of displaying β-hemolysis on blood agar and this distinguish it from the remaining organisms. They are Catalase negative. They cause various human diseases related to skin infections and other systemic diseases encompassing pharyngitis, erysipelas, cellulitis, necrotizing fasciitis. This bacterium is capable of producing toxins and may cause scarlet fever (Rayan, 2004). This organism possess various virulence factors which play an important role in its adhesion to the person’s tissues and therefore enable them to cross all the immunological barriers (Patterson, 1996). Citrobacter freundii is a gram-negative coliform bacteria. As the name suggest they are dependent on the citrate for their carbon source. They are capable of fermenting lactose and convert tryptophan to indole. They are omnipresent, in soil, water and waste. They seldom cause UTI and infant meningitis and sepsis (Drelichman, 1985). Klebsiella pneumoniae it is gram-negative bacterium and do not show any kind of motility, they possess capsule and are capable of fermenting lactose. They are present as the normal flora of mouth, skin and intestine (Rayan, 2004). It is capable of fixing nitrogen under anaerobic conditions. They do not show any indole production. In many aspects as all these are food/ water borne diseases and show almost analogous clinical picture, it is therefore essential to perform laboratory diagnosis for these severe forms and for the onset of medication. Ignorance can formulate the conditions to take fatal forms. From the above discussion we can conclude that safety measures are very crucial when consuming the food articles and water while travelling or on excursions. Furthermore, to prevent relapses proper and complete medication should be taken to prevent the causal organism develop resistance and to war the infection. Literature cited 1. Amyes, S., G. Enterococci and streptococci. Int. J. Antimicrob. Agents 29 Suppl 3: S43–52. 2007. 2. Drelichman, V; Band, J. D. "Bacteremias due to Citrobacter diversus and Citrobacter freundii. Incidence, risk factors, and clinical outcome". Archives of Internal Medicine 145: 1808–1810. 1985. 3. Messeley, K., Norrell, S., Microbiology Lab Manual. 2003. 4. Patterson, M. J. Streptococcus. In: Barons Medical Microbiology (Baron S et al., eds.) (4th ed.). Univ of Texas Medical Branch. 1996. 5. Ryan, K. J, Ray. C. G (editors). Sherris Medical Microbiology (4th ed.). McGraw Hill. 2004. Read More