Identification of the Unknown Microorganism - Lab Report Example

?Isolation and identification of the unknown micro organism Beneficial and harmful bacteria are present in all the food particles. Bacteriaproduce some biologically important molecules in the food and are thus beneficial to the human beings. Probiotics contain specific strains of bacteria that are useful for increasing and maintaining the gastrointestinal tract bacterial load. In the present study, the unknown microorganism present in the Probiotics, maozrella cheese and camembert cheese were analyzed using the biochemical tests such as catalase, oxidase, latex agglutination, Gram’s staining and string test. From the biochemical results it was identified that the unknown microorganisms present in the given samples of probiotics, mozeralla cheese and camembert cheese were Lactobacillus sp, Streptococcus sp and Penicillium sp. respectively. Introduction: Bacteria are ubiquitous in nature. They are beneficial and harmful to the human beings and animals. Bacteria are present in the food. Probiotics supplements contain specific strains of bacteria such as Lactobacillus and Bifidobacteria to increase the gut flora. Beneficial bacteria are present in the food and produce many important biological molecules. In the mozzarella cheese, Lactobacillus bulgaricus and Streptococcus thermophilus are present. Camembert cheese contains lactococcus and penicillium species are present. (Waites et al. 2009). Bacteria can be differentiated based on the enzymes secreted by them. Some enzymes are secreted out by the micro organisms enabling simple biochemical tests. The major exo enzymes present in bacteria are amylases, caseinase, gelatinase (hydrolytic enzymes), oxidase and catalase. (Gunasekaran 2007). Since most of the exoenzymes are hydrolytic enzymes which break down complex substances into simpler molecules, they are used as identifiers for the bacteria. (Sharma 2007). Catalase converts hydrogen peroxide into oxygen and water and oxidase oxidizes dichlorophenol indophenols from colorless to blue or red. (Benson 2001). The microorganisms present in the given samples can be isolated using the serial dilution technique and plated in the culture medium. The organisms are either gram positive or gram negative. The results obtained from the biochemical tests are used for the identification of the bacteria up to the genus level. (Reed et al. 2007). The number of bacteria present in the given sample is identified as the number of colony forming units. Colony forming units are then used to identify the number of bacteria present in the given diluted sample (Reed et al, 2007). The number of bacteria present in the given sample is calculated using the formula: Number of cells per gram = (Number of colonies (CFUs)) / (dilution x amount plated). The main objectives of this study are 1. Isolation of individual colonies from the given Probiotics, mozzarella cheese and camembert cheese samples. 2. Identification of the bacteria present in the given samples using the biochemical tests. Results: Table 1: food type and the morphological characteristics: Food type characteristics Probiotics 10-6 Circular, cream, raised and undulate Camembert 10-5 Pink, circular, convex and entire Camembert 10-4 Cream, punkiform , entire and convex Mozzarella 10-4 Brown, entire , circular Table 2: Colony count data Sample x10-3 x10-4 x10-5 x10-6 x10-7 probiotic - Greater than 300 Greater than 300 246 - Final count - 3 x 10^8 CFU / ml 3 x 10^9 CFU / ml 2.49 x 10^9 CFU / ml - Camembert 210 104 44 - - Final count 2.1 x 10^6 CFU /g 1.04 x 10^7 CFU / g 4.4 x 10^6 CFU / g - - Mozzarella 3 1 0 - - Final count 3 x 10^4 CFU / g 1x 10^5 CFU / g - - - Figure 1: The number of cells present in the given samples versus the dilution factor: Sample Colony characteristics Food characteristics Catalase Oxidase Gram stain String test Agglutination Probiotics 10-6 Soured , acidic Circular, cream, raised and undulate x x Positive (blue colored rod shaped cells) v X ( no agglutination was formed ) Probiotics 10-5, Soured , acidic Circular, cream, raised and undulate x x Positive (blue colored rod shaped cells) v X ( no agglutination was formed ) Mozzarella 10-4 Spongy, no smell, maintains it shape Brown, entire , circular x x Positive ( entire circular cells) v X ( no agglutination was formed ) Camembert 10-5 Strong smell, dense, semi solid Pink, circular, convex and entire x x X ( No results were produced) x X ( no agglutination was formed ) Control used E.coli : negative Staphylococcus :positive E.faecaus : negative E.coli: negative Staphylococcus positive ( blue color is produced ) Pseudomonas:positive ( blue color is produced ) Staphylococcus: gram positive, forms pair of clusters, circular E.coli: gram negative, rod shape. Staphylococcus: positive E.coli: forms string and negative Staphylococcus: positive (agglutination was formed ) E.coli: negative Table 3: Biochemical tests for the unknown microorganisms: Discussion: Bacteria are ubiquitous in nature. Bacteria are both beneficial and harmful. The bacteria present in the food either increases the value of the food or causes food spoilage. The fermentation is the process of adding micro organisms to the food and enabling them to produce some important biological molecules from them. The analysis of the micro organisms present in the given sample was performed using the biochemical tests. Oxidase, Catalase, Agglutination, Voges Prosker Test, Methyl red test, motility test, Gram’s staining are some of the biochemical tests used for the identification process. (Reed et al. 2007). In the present study, the Probiotics, camembert cheese and mozzarella cheese were analyzed for the micro organisms. The unknown bacteria are first grown in the broth and they are serially diluted at the dilution levels of 10-1 to 10-7. These tubes are then plated in the petriplates and kept for overnight incubation. The number of cells present in the given petriplates is identified using the colony forming units (CFUs). The number of colonies present in the given Probiotics for the dilution factors 10-4 , 10-5 and 10-6 are greater than 300, more than 300 and 246 colonies respectively. Similarly the number of colonies in Camembert cheese for the dilution factors 10-3 , 10-4 and 10-5 are 210, 104 and 44 respectively. The number of colonies present in the Mozarella cheese for the dilution factors 10-3, 10-4 and 10-5 are 3, 1 and 0 respectively. The number of bacteria present in the each plate is determined from the colony forming units using the formula: Number of cells per gram = (Number of colonies (CFUs)) / (dilution x amount plated). The probiotic sample had the bacterial load as 3 x 10^8 CFU / ml, 3 x 10^9 CFU / ml and 2.49 x 10^9 CFU / ml for the dilution factors 10-4 , 10-5 and 10-6 respectively. The camembert cheese had the bacterial load as 2.1 x 10^6 CFU /g, 1.04 x 10^7 CFU / g and 4.4 x 10^6 CFU / g for the dilution factors 10-3 , 10-4 and 10-5 respectively. Mozzarella cheese had the bacterial load as 3 x 10^4 CFU / g, 1x 10^5 CFU / g and 0 CFU/ g for the dilution factors 10-3 , 10-4 and 10-5 respectively. The Graph also represents the same. The number of bacteria was in the ratio of decreasing order as the dilution factor increased. The Probiotics had a high concentration of bacteria than mozzarella cheese and Camembert cheese. The colony count of the bacteria was greater than 300 for Probiotics but in mozzarella it was only 3. This indicates that bacterial load is very less in the mozzarella cheese. In the camembert cheese the bacterial load decreased gradually as the dilution factor increased. The overnight culture was then analyzed for the unknown microorganisms using the biochemical tests. Oxidase test, catalase test, Gram’s staining, latex agglutination test and string test were performed. It was observed that Probiotics showed positive for Gram’s staining and string test and negative for oxidase, catalase and agglutination test. (Neidhardt et al. 1990). Similarly the mozzarella cheese showed positive for Gram’s staining and string test and negative for oxidase, catalase and agglutination test. Camembert cheese showed negative doe all the tests. According to Ahmed and Kanwal paper, the unknown bacterium present in the probiotic was Lactobacillus sp. Lactobacillus sp is a rod shaped bacteria, form string and is gram positive. They show negative result for oxidase and catalase tests. (Ahmed and Kanwal 2004). Lactobacillus bulgaricus and Streptococcus thermophilus are the most dominant bacteria present in the mozzarella cheese. (Saxena and Awasthi 2003). Of these Lactobacillus is a rod shaped bacteria and Streptococcus sp is circular. Streptococcus sp shows catalase, oxidase and agglutination negative and positive for Gram’s positive and String test. In the experiment, we observed brown colored circular colonies that are Gram positive and String test positive. (Hoque et al. 2010). They were also negative for catalase test and oxidase test and latex agglutination test turned negative. From Dart‘s paper, the characteristics of the colony matches with the characteristics of Streptococcus sp. So Mozzarella cheese may contain Streptococcus sp. (Dart 1996). In the Camembert cheese, all the biochemical test results were negative and they did not undergo Gram’s staining. This indicates that bacterial species is not present in the medium and fungal species may be present in the camembert cheese. According to Lessard et al study, camcembert cheese contains penicillium species and even a fungal strain was named after camcembert, as Penicillium camembertii, so Penicillium sp must be present in the Camembert cheese. (Lessard et al. 2012). References: Ahmed, T and Kanwal, R., 2004. Biochemical Characteristics of Lactic acid producing Bacteria and preparation of Camel Milk Cheese by using Starter Culture, Pakistan Veterinary Journal, Vol.24, No.2. Benson, HJ., 2001. Microbiological Application: Laboratory Manual in General Microbiology, McGraw –Hill. Dart, RK., 1996. Microbiology for the Analytical Chemist, Royal Society of Chemistry. Gunasekaran, P., 2007. Laboratory Manual in Microbiology, New Age International. Hoque, MZ, Akter, F, Hossain, KM, Rahman, MSM, Billah, MM and Islam, KMD., 2010. Isolation, Identification and Analysis of Probiotic Properties of Lactobacillus Spp. From Selective Regional Yoghurts, World Journal of Dairy and Food sciences, Vol.5, No.1, pp: 39-46. Lessard, MH, Belanger, G, St- Gelais, D and Labrie, S., 2012. The Composition of Camembert Cheese-ripening Cultures modulates both Mycelia Growth and Appearance, Applied and Environmental Microbiology, Vol.78, No.6, pp: 1813- 1819. Neidhardt, FC, Ingraham, JL, and Schaechter, M., 1990. Physiology of the Bacterial Cell: a Molecular Approach, Sinauer Associates. Reed, R, Holmes, D, Weyers, J and Jones, A., 2007. Practical skills in Biomolecular Sciences, Pearson Education. Saxena, NP and Awasthi, DK., 2003. Krishna’s Microbiology, Krishna Prakashan Media P Ltd. Sharma, K., 2007. Manual of Microbiology, 2nd edition, Ane books Pvt ltd. Waites, MJ, Morgan, NL, Rockey, JS and Highton, G., 2009. Industrial Microbiology, John Wiley and Sons. Read More