Lab Report Microbiology Essay Example for Free

Lab Report Microbiology EssayAbstractdairy nourishment staff such as soft cheese,cream cheese,raw take out,sour cream, yoghurt and probiotic yoghurt products can be a rich source of diverse lactic window glass bacterium.The objective of this lab practical was to isolate lactic acid bacteria(LAB) haoma raw milk,establishment of pure cultures of LAB,identify LAB and phage recovery and enumeration of recoverd phage.Raw milk was chosen as a sample so as to have a more corroborative result.To identify bacteria Lab isolated from raw milk,biochemical,morphological ,physiological and cultural characteristics were employed. The purification of isolates was done by moving Gram +ve ro ds and coccus shaped bacteria to selective media MRS and M-17 plates. The isolates were sub cultured till pure isolates were got. From 20 raw milk samples a total of 150 LAB positives were got, in which 22 and 128 were identified as lactic acid cocci and lactic acid bacilli,respectively.Also, our bioch emical interrogatorys showed the occurrence 11 and 13 of 11 and 13 Lactococcus lactis subsp. cremoris and Leuconostoc mesenteroides subsp. cremoris among lactic acid cocci.In the case of lactic acid bacilli, Lactobacillus helveticus 18 Lactobacillus plantarum 37 Lactobacillus brevis 8 Lactobacillus casei subsp. casei 18 and Lactobacillus delactobacillirueckii subsp. bulgaricus 47 was found. In the lactic acid cocci and bacilli, Leuconostoc mesenteroides subsp. cremoris and Lactobacillus delactobacillirueckii subsp. bulgaricus were found to be the more dominant species, respectively.Bacterial phages were inducted from the Lactic acid bacteria and enumerated by utilize several biochemical techniques.INTRODUCTIONTo produce flavor and acidity at coveted levels,fermented milk products argon prepared in controlled fermentation of milk. (Thapa, 2000). Starter culture organisms in this fermentations be spaciouss to bacteria family known as the Lactic Acid Bacteria (LAB). These LABS are i dentified by of morphological,and physiological characteristics.LAB are widely found in nature and almost in all micro flora.LAB are gram positive bacteria and are important in food fermentation. Other species of the genus Lactobacillus, Lactococcus and Leuconostoc are added to this group. The lactic acidfermentation process has been known by human for long time and even applied in some activities.LAB has also been an efficient method of natural preservation.Furthermore lab determine the nutritional value,flavor and texture of food and feeds). Industrialization of the biological revolution of foodstuffs has LAB an ecomonic boost beca function they are important in safety aspects of fermented products. Lactic acid is used by food sedulousness as an acidulent and preservative for the production of sour curd cheese and yoghurt ( tie beamater and Griffin, 1971). Lactococci are the major mesophilic bacteria used for acid production in dairy fermentations and used as starter cultures in the manufacture of a vast range of dairy foods including fermented milks, lactic butter, cheese and lactic casein (Ward et al., 2002).MATERIALS AND METHODSRaw milk samples Raw milk samples were collected in sterilized specimen bottles from the local dairy shops around the university,including the raw milk from the universitys dairy department.The raw milk were kept at 4 for more use. lactic acid bacteria isolation from raw milk The samples were weighed and homogenized aseptically.Each sample, a 1.10 dilution was made by apply peptone water and then by making a 10 pack of continued dilution. The 0.1 ml taken from each dilution was then sub cultured duplicately into the M 17 and MRS nutritious nutrient agars used for isolating LAB (Badis et al., 2004a Guessas and Kihal, 2004).In order to counter yeast growth the media were then added with 100 mg of cyclo-heximide prior to being incubated in best temperatures ( 30C) for 3 days (Beukes et al., 2001 Kalavrouzioti et al., 2005).The ag ar plates of MSR were incubated in anaerobic conditions using the Gas-Pack system at 30C for 3 days to provide an optimum temperature for outgrowth the different genus of bacteria. M17 agar plates were also incubated in anaerobic conditions at 30C for 2 days to set up an optimal temperature for growing lactococci.Higher dilutions were used to perform total counts. Colonies were then selected randomly and the streak plating method employed to purify the stains. The strains were kept in 2 conditions including at 4C (for MRS and M17 plates ) and at -20C (for M17 and MRS broth) withh by 20% glycerol.Identification of the bacterial strainsThe strains were subjected to gram staining,catalase and spore formation tests. (Harrigan and McCance, 1976).All Colonies were characterized in MRS and M 17 agars.The strains that gave gram positive and catalse oppose results were set aside for further identification.(Sharpe, 1979).The growth of the bacteria at different temperatures of amongst Growt h 10-45C for 3-6 days , resistance to 60C for 30 min (Sherman test), growth in the presence of 2 to 6 % NaCl and different pHs (4.5 and 6.5) were used to identify the strains of LAB. Arginine and asculin hydrolysis,citrate utilistaion, acetone productionformation of gas from glucose and production of dextran from sucrose were also determined. The starins were then tested for fermentation of L-arabinose, D-xylose, galactose, D-fructose, sorbitol, lactose, melibiose, saccharose, D-raffinose, melezitose, mannose and glucose.Bacterial growth in the different temperatures were confirmed by turbidity change in MRS or M17 after incubation(after 24,48 and 72 hrs).Microbial allowance to the diverse levels of salt, pH and heat was evaluated. Arginine dihydrolase agar and asculin acid agar were used to perform the hydrolysis tests. For determination of citrate utilization and acetone production, citrate and MR-VP agars were used. MRS or M17 broths with shorthorn renders were used for determi nation of gas production and the detrin production from sucrose was done in MSR.To assess the sugars fermentation in a medium a response with the following composition was used (gL-1) bovine extract, 10.0 neopepton, 10.0 yeast extract, 5.0 K2HPO4, 2.0 CH3COONa+3H2O, 5.0 diamonium citrate, 2.0 MgSO4, 0.2 MnSO4, 0.05 brom-cresol-purple, 0.17 tween 80, 1 mL. Carbon utilization was also tested.Phage inductionMRSA broth liquid culutures were equally shared into two sterile tubes.Each tube was labeled as mitomycin C and the other as control. 500l of micomycin was added to the tube labelld as mitomycin and ascpetic techniques of flaming the neck before and after adding the mycomycin. A starch agar plate marked STA containing nutrient agr with soluble starch was already provided.A casein agar plate that contained nutrient agar mixture added skim milk was given and marked CA. All the three plates were inoculated by streaking of the MRSA Lactobacillus lattis culture. This wasdone with the h elp of the loop. The loop was flamed and a colony of the culture was collected. The plates were then streaked with the culture. The plates were then incubated for 12-18 hours at 37oC. The bacteria were also transferred into the nutrient agar plate to set up for biochemical tests.. reckoning of bacterial phagesPhage stock was diluted to achieve a plaque count on plates of 100-250 pfu (plaque forming units).All the dilutions were mixed thoroughly in a sterile saline. The phage was then plated by removing one soft agar at a time,then adding 0.3ml of bacterial suspension to it.This was also followed by 0.1 of diluted phage The agar tube was rolled between palms to mix and quickly pour to suface of warm stalk agar plate. Quick gentle figure patterns were done on the surface of the base plate agar The agar was allowed to harden and incubated for 35 degree celcious for 8 hours.ResultsCatalase testAfter incubation, hydrogen peroxide was added to the one colony on the nutrient agar plate. S mall bubbles of oxygen wereformed which indicated a positive result for catalase.Figure 2 The catalase testStarch hydrolysis testWhen iodine solution was poured to the starch agar plate and allowed to rest for close to 2 minutes,the plate turned blue which indicated the presence of starch that has not been hydrolysed.A BFig 1 -Growth of MRSA Lactobacillus lattis on starch agar plate (A) before the addition of iodine solution and (B) after the addition of iodine solution.Agar testThe position of the growth in the tube was observed. The growth was throughout the tube, but near the surface, the growth was highest whichindicated being aerotolerant.Figure 3- The bacteria stabbed in both the tubes containing NA and MRS.Carbohydrate fermentation substratesAPI test strips were used to identify the bacteria and the results showed it was Lactococcus lactis sspcremoris 1.Casein HydrolysisThe casein agar plates were examined to see any clearing around the colonies after being incubated for 48 hours. There was no clearing of the agar around the bacterial growth. Therefore, the results showed negative casein hydrolysis.Gelatin hydrolysisSaturated ammonium sulpahate was added onto the gelatine agar plate there was no temerity indicating negative hydrolysisFigure 4 The results obtained after the data was entered on the computer database.Figure 5 The difference between a control and the samples of bacteria.Test for phage inductionOnce mitomycin C was added to the MRS liquid broth, it was observed for the induction of phages. It showed there was a clear lysis of the turbid culture.Figure 6 Comparison between a control and bacteria culture containing mitomycin CAll 150 Gram +non-sporeforming ans catalase negative were charcterised as followsMesophilic homo-fermentative cocci, 11 isolatedIt was characterized byarginine dihydrolase negative, arginine hydrolysis negative, citrate negative and acetoin negative This gropu was identifies as Lactococcus lactis subsp. Cremoris .The microorganisms were spherically shape.They occurred in pairs with non motile, facultative anaerobic fermentative metabolism. Mesophilic heterofermentative cocci, 13 isolatedMicroorganisms in this group had a close coition with Leuconostoc mesenteroides subsp. cremoris .They were arginine negative,glucose positive,acetonoine positive and dextrane positive. Lactobacilli bacteria, 128 isolated The group was divided into 3 (1) Mesophilic facultative heterofermentative Lactobacilli (55 isolates)Included Lactobacilli plantarum (37 isolates)and Lactobacilli. casei subsp. casei (36 isolates, (2) Thermophilic obligate homo-fermentative Lactobacilli (64 isolates) Included Lactobacilli. helveticus (17 isolates) and Lactobacilli. delactobacillirueckii subsp. bulgaricus (47 isolates). They were lactose positive andfructose positive.(3) mesophilic obligate hetero-fermentative Lactobacilli (8 isolates) Included Lactobacilli. brevis (18 isolates)DiscussionIt was discovered that mesophilic faculta tive hetero-fermantative lactobacilli group was divided into two37 isolates were identified to be lactobacilli plantarum ans 18 isolates as lactobacilli casei subsp.casei.This results are also consistent with other research working such as the isolation of lactic acid bacteria from Maasai handed-down fermented milk(Mathara et al.,2004). For the second group,17 isolates were identified as Lactobacilli plantarum and 47 isolates identified as lactobacilli delactobacillirueskii subs.bulgaricus. Furthermore,lactobacilli brevis isolates(8) were identified using mannose and melezitose fermentation. In the cocci group,12 and 22 isolates were identified as Leuconostoc mesenteroides subsp. cremoris and Lactococcus lactis subsp. cremoris. Respectively.This number is low an its attributed to the fact lactic acid cocci are not able to struggle with lactic acid bacilli in mixed cultures(Teuber and Geis, 1981 Togo et al., 2002).LAB are presenta in dairy manufacturing as starter cultures.There a re specific fermentation processes that have been true to maximize the growth of desired LABspecies.Some of the species are fastidious organisms like Lactobacilli delactobacillirueckii subsp. bulgaricus and Lactobacilli helveticus (Bottazzi, 1988). Isolates that belong to lactobacilli plantarum group are shown to be dominant members of LAB flora of acid fermented stuff(tempoyuk).Morever, Lactobacilli. brevis group and Ln. mesenteroides isolates were also found (Leisner et al., 2001).This isolates have alos been found in South African Traditional fermeneted products.There are also other isolates that have been found in raw gaots milk of Algerian origin.This species include Lactobacilli. helveticus, Lactobacilli. plantarum, Lactobacilli. delactobacillirueckii subsp. bulgaricus, Lactobacilli. brevis and Lc. lactis subsp. lactis (Badis et al., 2004b). In chili bo, Lactobacilli. plantarum isolates were found to be the most dominant organism(Leisner et al. 1999).REFERENCESAccolas, J.P. a nd J. Auclair, 1977. Determination of the acid producing activity of intemperate frozen suspensions of lactic acid bacteria. Lait, 50 609-626.Ammor, S., C. Rachman, S. Chaillou, H. Prevost and X. Dousset et al., 2005. Phenotypic and genotypic identification of lactic acid bacteria isolated from a small-scale facility producing traditional dry sausages. Food Microbiol., 22 373-382. CrossRef Badis, A., D. Guetarni, B. Moussa-Boudjema, D.E. Henni and M. Kihal, 2004. Identification and technological properties of lactic acid bacteria isolated from raw goats milk of four Algerian races. Food Microbiol., 21 579-588. CrossRef Badis, A., D. Guetarni, B. Moussa-Boudjema, D.E. Henni, M.E. Tornadijo and M. Kihal, 2004. Identification of cultivable lactic acid bacteria isolated from Algerian raw goats milk and evaluation of their technological properties. Food Microbiol., 21 343-349. CrossRef Beukes, E.M., B.H. Bester and J.F. Mostert, 2001. The microbiology of SouthAfrican traditional ferment ed milks. Int. J. Food Microbiol., 63 189-197. CrossRef PubMed Direct contact lens Bottazzi, V., 1988. An introduction to rod shaped lactic-acid bacteria. Biochemie, 70 303-315. PubMed Collins, M.D., B.A. Phillips and P. Zanoni, 1989. Deoxyribonucleic acid homology studies of Lactobacillus casei, Lactobacillus paracasei sp. nov., subsp. paracasei and subsp. Tolerans and Lactobacillus rhamnosus sp. nov., comb. nov. Int. J. Syst. Bacteriol., 39 105-108. CrossRef Guessas, B. and M. Kihal, 2004. Characterization of lactic acid bacteria isolated from Algerian arid zone raw goats milk. Afr. J. Biotechnol., 3 339-342. Direct Link Harrigan, W.F. and M.E. MaCance, 1976. Laboratory Methods in Food and Dairy Microbiology. Revised Edn., Academic Press, freshly York, pp 33-200.Hemme, D. and C. Foucaud-Scheunemann, 2004. Leuconostoc, characteristics, use in dairy technology and prospects in functional foods. Int. Dairy J., 14 467-494. CrossRef Herrero, M., B. Mayo, B. Gonzalez and J.E. Suarez , 1996. Evaluation of technologically important traits in lactic acid bacteria isolated from spontaneous fermentation. J. Applied Bacteriol., 82 565-570. Direct Link Holt, J.G., 1994. Bergeys Manual of Determinative Bacteriology. 9th Edn., Williams and Wilkins, Baltimore, Pages 787.Kalavrouzioti, I., M. Hatzikamari, E. Litopoulou-Tzanetaki and N. Tzanetakis, 2005. Production of hard cheese from caprine milk by the use of two types of probiotic cultures as adjuncts. Int. J. Dairy Technol., 58 30-38. CrossRef Lee, B., 1996. Bacteria- Based Processes and Products. In Fundamentals of Food Biotechnology, Lee, B. (Ed.), Wiley-Inter Science, New York, pp 219-290.Leisner, J.J., B. Pot, H. Christensen, G. Rusul and J.O. Olsen et al., 1999. Identification of lactic acid bacteria from Chilli Bo, a Malaysian food ingredient. Applied Environ. Microbiol., 65 599-605. PubMed Leisner, J.J., M. Vancanneyt, G. Rusul, B. Pot, K. Lefebvre, A. Fresi and L.K. Tee, 2001. Identification of lactic acid bact eria constituting the predominating microflora in an acid-fermented condiment (Tempoyak) popular in Malaysia. Int. J. Food Microbiol., 63 149-157.Linkater, P.M. and C.J. Griffin, 1971. Immobilized living jail cell fermentation. J. Dairy Res., 38 127-136.Lipinsky, E.S., 1981. Growth and activity of Lactococcus lactis ssp. cremoris in skim milk. J. Sci., 212 1465-1471.Mathara, J.M., U. Schillinger, P.M. Kutima, S.K. Mbugua and W.H. Holzapfel, 2004. Isolation, identification and characterization of the dominant microorganisms of kule naoto The Maasai traditional fermented milk in Kenya. Int. J. Food Microbiol., 94 267-278. PubMed Mayeux, J.V., W.W.E. Sandine and P.R. Elliker, 1962. A selective medium for detecting Leuconostoc organisms in mixed strain starter cultures. J. Dairy Sci., 45 655-656.Muyanja, C.M.B.K., J.A. Narvhus, J. Treimo and T. Langsrud, 2003. Isolation, characterization and identification of lactic acid bacteria from bushera A Ugandan traditional fermented beverage. I nt. J. Food Microbiol., 80 201-210. CrossRef Olarte, C., S. Sanz, E. Gonzalez- Fandos and P. Torre, 2000. The effect of a commercial starter culture addition on the ripening of an artisanal goatscheese (Cameros Cheese). J. Applied Microbiol., 88 421-429. PubMed Samelis, J., F. Maurogenakis and J. Metaxopoulos, 1994. Characterization of lactic acid bacteria isolated from naturally fermented Greek dry salami. Int. J. Food Microbiol., 23 179-196. PubMed Server-Busson, C., C. Foucaud and J.Y. Leveau, 1999. Selection of dairy Leuconostoc isolates for important technological properties. J. Dairy Res., 66 245-256. CrossRef Sharpe, M.E., 1979. Identification of the Lactic Acid Bacteria. In Identification Methods for Microbiologists, Skinner, F.A. and D.W. Lovelock (Eds.). Academic Press, London, pp 233-259.Stiles, M.E. and W.H. Holzapfel, 1997. Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol., 36 1-29. Direct Link Terzic-Vidojevic, A., M. Vukasinovic, K. Vel jovic, M. Ostojic and L. Topisirovic, 2007. Characterization of microflora in homemade Int. J. Food Microbiol., 114 36-42. PubMed Teuber, M. and A. Geis, 1981. The Family Streptococaceae (Non-Medical Aspect). In The Prokaryotes A Handbook on Habitats, Isolation and Identification of Bacteria, Starr, M.P., H. Stolp, H.G. Trueper, A. Balows and H.G. Schlegel (Eds.). Springer-Verlag, Berlin, pp 1614-1630.Thapa, T., 2000. Small-Scale milk Processing Technologies Other Milk Products. FAO, Rome, Italy.Togo, C.A., S.B. Feresu and A.N. Mutukumira, 2002. Identification of lactic acid bacteria isolated from Opaque beer (Chibuku) for potential use as a starter culture. J. Food Technol. Afr., 7 93-97. Direct Link Tserovska, L., S. Stefanova and T. Yordanova, 2002. Identification of lactic acid bacteria isolated from Katyk, goats milk and Cheese. J. Cult. Collect.,3 48-52. Direct Link Ward, L.J.H., G.P. Davey, H.A. Heap and W.J. Kelly, 2002. Lactococcus Lactis. In Encyclopedia of Dairy Science, Roginski, H., J.W. Fuquay and P.F. Fox (Eds.). Elsevier Sci. Ltd., London, pp 1511-1516.