The purpose of the experiment is to determine the microbilogical effects over the given food sample and to analyze the samples for coliforms using the traditional Most Probable Number (MPN) method in Lauryl Sulfate Tryptose broth to indicate the overall rate of contamination .Spoilage organisms pseudomonads in pseudomonas selective agar and the E.coli are determined by subculturing positive MPN tubes.The series of pourplate and spread plate techniques were employed for the given samples.Streaking was done for e.coli, pseudomonas fluorescens and salmonella in the selective agars.The quality and their shell life were determined from the results obtained.
One of the common microbiological tests carried out in food is the total viable count which is also known as the standard plate count or aerobic plate count.In this, suitable dilutions of the food sample are plated on or in agar based media containing complex nutrients which support the growth of as wide a range of microorganisms as possible .Nutrients included in,for example nutrient agar (oxoid) are beef extract,yeast extract and peptone (a proteolytic enzyme digest of fresah meat containg a variety of inorganic salts, growth factors and peptides.)The Ph of the medium is usually adjusted to 7.0-7.4 so that bacteria rather than yeasts or moulds are recovered.
The individual bacterial cells transferred to the plate in the diluents divide in the normal way during incubation.Thus an estimate of the total number of viable cells (i.e.,.cells capable of growth in the recovery medium) in the dilution plated out can be calculated by counting the total number of bacterial colonies which develop following incubation; clearly,under normal aerobic conditions obligate aerobes and facultative anaerobes will grow (Palleroni, N.J. (1984).
The incubation temperatures selected depend upon on the food being examined.Commanly used temperatures are 55°c for thermophiles, 35-37°c for mesophiles and 20-30°c for any spoilage bacteria.Whilst the latter temperatures are suitable for most psychrotrophs.It miust always be borne in mind that no one incubation temperature completely excludes all organisms from another group.
Many plating techniques are employed for enumerating total numbers of viable bacteria and three are described briefly.Enumeration of colonies is traditionally performed manually using an illuminated colony counter with the operator counting each individual colony.This can be tedious operation and ,unless a suitable number of colonies has developed in the growth medium (ideally below 300,although for statistical reasons a minimum of 30 colonies is also required ),it can also be inaccurate.In the recent years automatic colony counting devices have been developed which enable accuratecounts to be obtained in a few seconds.
(Food hygiene microbiology and HACCP by S.J.FORSYTHE,P.R. HAYES) .The pour plate technique can be used to determine the number of microbes/mL or microbes/gram in a specimen.
Vegetative cell is an ordinary bacterial cell under normal condition which constantly working to live, grow and reproduce. But vegetative cells are vulnerable to damage from many environmental forces.Under drastic condition forms endospores and this results in spore formation.At this state the cells are inactive and dormant. They produce vegetative cells in return of favourable conditions.
Each colony represents a "colony forming unit" (CFU). For optimum accuracy of a count, the preferred range for total CFU/plate is between 30 to 300 colonies/plate.
One disadvantage of pour plates is that embedded colonies will be much smaller than those which happen to be on the surface, and must be carefully scored so that none are overlooked. Also, obligate aerobes may grow poorly if deeply imbedded in the agar. MostE. colistrainsare harmless, but some, such asserotypeO157:H7, can cause seriousfood poisoninginhumans, and are occasionally responsible forproduct recalls.The harmless strains are part of thenormal floraof thegut, and can benefit their hosts by producingvitamin K2,or by preventing the establishment ofpathogenicbacteria within the intestine.
pseudomonasis agenusof gammaproteobacteria, belonging to the larger family ofpseudomonads. Other characteristics which tend to be associated withPseudomonasspecies (with some exceptions) include secretion of pyoverdin (fluorescein), afluorescentyellow-greensiderophoreunder iron-limiting conditions. CertainPseudomonasspecies may also produce additional types of siderophore, such as pyocyanin byPseudomonas aeruginosaand thioquinolobactin byPseudomonas fluorescens,.Pseudomonasspecies also typically give a positive result to the oxidase test, the absence of gas formation from glucose, glucose is oxidised in oxidation/fermentation test using Hugh and Leifson O/F test, betahemolytic(on blood agar),indolenegative,methyl rednegative,Voges Proskauertest negative, citrate positive.
The genus demonstrates a great deal ofmetabolicdiversity, and consequently are able to colonise a wide range of niches. Their ease of culturein vitroand availability of an increasing number ofPseudomonasstraingenomesequences has made the genus an excellent focus for scientific research; the best studied species includeP. aeruginosain its role as an opportunistic human pathogen, the plant pathogenP. syringae, the soil bacteriumP. putida, and the plant growth promotingP. fluorescens. Reid G, Howard J, Gan BS (September 2001).
MATERIALS AND METHODS:
Recieved beef samples and bean sprouts wrer noted for product information.Then they were serial diluted upto 10^-6 dilutions. 10^-4, 10^-5 and the 10^-6 dilutions of both beef and bean sample were pour plated in the PLATE COUNT AGAR (PCA).Similarly the dilutions 10^-1, 10^-2, 10^-3 of both samples were spread plated in Pseudomonas selective agar. Streaking is done for E.coli, salmonella and Pseudomonas fluorescens in pseudomonas selective agar.E.coli is streaked in Endo agar. The dilutions 10^-3, 10^-4, 10^-5 and the 10^-6 of beef sample were transferred (100ml) to 5 test tubes.Each dilutions in 5 lauryl sulphate tryptose tubes totally 20 test tubes. Similarly the same is done for bean sample.
Plate countagar(PCA) is a microbiologicalgrowth mediumcommonly used to assess or to monitor "total" or viable bacterial growth (E.coli) of a sample. PCA is not a selective medium.The composition of plate count agar may vary.So , when both samples are pour plated in this agar no colony formation was reported for beef sample.Whereas, the 62 * 10^6 cfu/ml was reported in the dilution 10^-6 in bean sample..Since microorganism in foods represents various populations with many different growth requirements ,the optimum conditions for determining the total viable count may vary from one food to another.The conventional aerobic plate count for examining the beef and bean sample is subjected to PCA.Generally,the microorganism showed delayed or no growth formation in the selective agar for beef sample.However they showed aggressive or favoured growth rate for bean sample.The E.coli which is found to be an important spoiler in these type are found TNTC for dilutions10^-4 , 10^-5.
Similarly, In spread plate technique no colony formation is reported for the beef sample in the pseudomonas selective agar.While TNTC is reported for all dilutions in bean sample.Specific microbiological technique for confirmation of presumptive isolates of pseudomonas.spp are of great importance since food and environmental samples often contain a complex microflora which can interfere with isolation and confuse identification.Therefore,it seems to be recommendale for isolation in bean sample.They usually produce a colonies with blue green or brown pigment .
The dilutions 10^-3,10^-4,10^-5,10^-6 were transferred to tubes containing lauryl sulphate tryptose.This methoid actively determines the most probable number for each sample in different dilutions.The positive result was seen in bean sample .For 10^-3 dilution 3 test tube is found ,while 2 proved positive in 10^-4 dilution and 1 proved positive in 10^-5 dilution.However,no positive result is obtained for ground hamburger.They are tabulated based on the assumption of a poisson distribution of coliforms in the sample. A positive LST tube contains an air bubble trapped in the inverted Durham tube. Convert the number of colonies into presumptive coliforms per g found in the original sample.
Streaking is done for E.coli, salmonella and Pseudomonas fluorescens in pseudomonas selective agar.E.coli is streaked in Endo agar. The streaked E.coli turned the light brown coloured agar (brilliant green agar) to light green. And the colonies formed on the endo green agar were golden green, smooth and spherical shaped. Because these agar have substantial lactose,sodium sulphite and basic fuchsin.
Salmonella species plated, turned brilliant green agar into red colour with pink coloured colonies. The purpose of this step is to promote salmonella bacteria population and facilitate their growth presence. Bacteria from the enrichment broth are subsequently sub cultured by streaking out on a selective agar substrate such as BG agar. While the colonies of pseudomonas species plated on brilliant green agar were whitish and smooth.
Experiment is done in order to determine the bacterial enumeration which showed that beans sample is heavily contaminated.Whereas,hamburger is found to be free from such spoilage and exhibited increased shell life and spoilage.MPn showed the value of 3.09 x 104 bacteria/g. The streaked E.coli turned the light brown coloured agar (brilliant green agar) to light green. And the colonies formed on the endo green agar were golden green, smooth and spherical shaped. While the colonies of pseudomonas species plated on brilliant green agar were whitish and smooth. Salmonella species plated, turned brilliant green agar into red colour with pink coloured colonies.
- Reid G, Howard J, Gan BS (September 2001). "Can bacterial interference prevent infection?".Trends Microbiol.
- Palleroni, N.J. (1984) Pseudomonadaceae. Bergey's Manual of Systematic Bacteriology. Krieg, N. R. and Holt J. G. (editors) Baltimore: The Williams and Wilkins Co., pg. 141 - 199.
- Compeau, G., Al-Achi, B.J., Platsouka, E., and Levy, S.B. (1988) Survival of rifampicin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems. Appl. Environ. Microbiol.
- DeFlaun, M.F., Tanzer, A.S., McAteer, A.L., Marshall, B., and Levy, S.B. (1990) Development and characterization of an adhesion-deficient mutant of Pseudomonas fluorescens. Appl. Environ. Microbiol.