Isolation, Enumeration, Identification and Confirmation of Food-Poisoning Microbes from Food - Lab Report Example

Isolation, Enumeration, Identification and Confirmation of Food-Poisoning Microbes from Food (lab report) Food poisoning is a serious condition that can result in severe worsening of the health and wellbeing of people. Modern day life introduced fast food and fast meals that are composed of high calorie ingredients that are very good environment for proliferation of bacteria that can cause food poisoning. In our experiment we will have to identify the presence of bacteria that may cause a food born disease in lettuce. Lettuce can often be contaminated with different bacteria that can cause food born disease. Lettuce can be contaminated with dirt, fecal materials from the earth where is grown, with secretions from the people who manage the distribution and preparation of the food etc. In our experiment we will test 10g of lettuce for presence of bacteria that presumably caused outbreak of food-poisoning in a local school. Several children have been complaining of diarrhea, abdominal pain and some have started passing blood in their stool. The lettuce was on the menu as a fresh salad 3 days earlier. We will try to conduct microbiological investigation of the bacteria that are present in the lettuce. Then we will identify if there are present any organisms that can cause food born infection. If organisms are found we will try to find the specific group of bacteria. Also we will have to calculate the concentration of these bacteria and made a conclusion if this concentration is sufficient to cause a disease. Results In order to test for presence for bacteria we will prepare the food sample for examination. The lettuce was placed in Maximum recovery diluent (MRD) which is an isotonic liquid that contains small concentrations of peptone and it is used to maintaining the viability of eventually present organisms in the food. We than placed the lettuce in 90ml of MRD and put it in the stomacher for 30 sec. The stomacher transforms the food sample into homogenous suspension and promotes the releasing the bacteria into the liquid medium (MRD). We than made different dilutions of the suspension (10-1 10-2 10-3 10-4 10-5 and 10-6) and inoculated them on different agar mediums. We used: - Nutrien agar (NA) which is unselective medium for growth. And will help us to identify the total number of bacteria present. - Violet Red Bile Glucose agar (VRBG) which will help us to differentiate the number of bacteria belonging to the enterobacteriace species (Escherichia spp., Shigella spp., Slmonella spp. Etc) - Sorbitol MacConkey agar (SMAC) which is medium for selective growth of E. Coli bacteria. - Xylose Lysine Deoxycholate agar (XLD) which is selective medium for growing of Salmonella and Shigella species. The plates were incubated for 24 hours on 37 degrees Celsius. After this perioed we examined the plates. Nutrition agar that was inoculated with 10-6 dilution of the suspension had 196 colony forming units, and according from the provided guidance, this dilution was used to calculate the colony forming units per gram according to the formula cfu/gram = No. of colonies x 10 x dilution factor. According to the formula the result nutrient agar produced 10140 cfu/gram. The Violet red bile glucose agar (VRBG) that was inoculated with dilution of 10-6 produced 1040 colonies and according to the formula we calculated that had concentration of bacteria of 62400 cgu/gram. Since VRBG agar is selective we can presumably conclude that these colony forming units belong to enterobactericeae family. The sorbitol MacConkey agar (SMAC) that was inoculated with dilution of 10-6 of the suspension produced 200 colonies, and therefore we calculated that had concentration of 12000 cfu/gram. Since this SMAC agar is also selective we can conclude that these were enterohemorrhagic  E. Coli bacteria. The Xylose lysine deoxycholate agar (XLD) that was inoculated with dilution of 10-4 produced 76 colonies and based on the above mentioned equation the concentration of bacteria was 3040 cfu/gram. Since this agar is specific and promotes growth of Salmonella and Shigella species we can conclude that most of the bacteria are from this species. Since we didn’t see any red colonies with black center which is specific for Salmonela species, we can presumably conclude that the bacteria isolated were from the Shigela species. As we can see selective agars produced significant number of colonies. However even though they are selective we must conduct additional confirmation test in order to be sure in the positive identification of the bacteria. There is no need to conduct confirmatory tests on colonies on nutrient agar and Violet Red Bile Glucose agar because they are less selective and there are different types of bacteria present. In order to confirm that bacteria isolated on SMAC agar are E. Coli we done additional oxidase test. One colony of the SMAC agar was inoculated on Nutrient agar and incubated 24 hours on 37 degrees. We than used Oxidse test strip on one of the colonies and the result was oxidase negative. We took another colony and we streaked in on microscope glass and then we allayed Gram staining. We examined the microscopic glass under a microscope and we identified Gram negative bacteria. In order to additionally confirm that the bacteria isolated on SMAC agar is E Coli we tested the bacteria using API20E bacterial identification strip. We used the procedure as described in the laboratory guide. We used one colony from the secondary nutrient agar and dispensed in saline solution. We filled all of the tubes of the bacteria identification strip as described in the laboratory guide and we incubated the strip for 24 hours on 37 degrees Celsius. We obtained the results from the identification strip and we received result 4044500 which we than used this result on the API web program and received E. Coli positive result. In order to better understand the oxidase test we done additional examination on plate inoculated with Bacillus cereus bacteria. We tested the bacteria and we get oxidase positive result with 99.6% accuracy. Discussion E Coli is Gram negative and oxidase negative, rod shaped bacteria. It is found as a normal inhabitant of the lower part of the intestines in mammals and humans. There are many subtypes of Echerichia Coli that are harmless and present as a normal flora in the intestine of humans, but some strains like serotype 0157 can cause severe gastroenteritis. E. Coli species can survive a short amount of time outside the body and the intestine tract and this is why if these bacteria are found in the food it is a sign of fecal contamination. Salmonella and Shigella are Gram negative rods that can be a cause of gastroenteritis. The presence of these bacteria in the food is also a sign of fecal contamination and measures should be taken to prevent infection if these bacteria are found in large quantities in the food. Shigella can be found as a normal flora in some humans and is widely present in other mammals and domestic animals. It can cause disease only in humans and other primates but not in other animals. Salmonella on the other hand is not part of the normal bacterial flora and if it is found it is a sign of infection. As we can see from the results from the plates we inoculated we produced different numbers of colonies. Nutrient agar produced 196 colonies. This can tell us about the microbiological load of the food. Nutrient agar is not selective for pathogenic bacteria and therefore can give us only qualitative information’s about the aerobic bacteria that are present in the food. If the microbial loading is very high we can conclude that the food was not well stored and that there is higher degree of contamination. Violet Red Bile Glucose agar (VRBG) on the other hand is more selective growth medium and promotes the growth of Enterobactericeae family of bacteria. This plate is more important in evaluating the results in our experiment because it can tell us if there is a contamination with pathogenic bacteria that can cause disease. It is less specific than Xylose Lysine Deoxycholate agar (XLD), but the bacteria that grow on this medium have the potential to cause food borne diseases. Using VRBG agar we calculated 62400 cgu/gram or 6.24 x 105 bacteria per gram. This is a sign of fecal contamination of the lettuce we tested. As we mentioned XLD agar is more selective medium for growing Salmonella and Shigella species and we didn’t detect any Salmonella colony forming unites in the plate, so we can conclude that presumably most of the bacteria are from the Shigella species. We detected 3040 cfu/gram or 3.04 x 103 cfu/gram of bacteria in the XLD agar. This is also a sign of fecal contamination. Based on these two plates we can conclude that the food was probably contaminated with fecal materials that were present in normal flora of mammalian organisms, because no Salmonela was detected. To further analyze the lettuce we inoculated the smple of SMAC agar and received 12000 cfu/gram or 1.2 x 104 cfu/gram concentration in the lettuce. This is also a sign of fecal contamination. But since the results that we obtained from the SMAC agar are only presumptive for E Coli we did additional test to confirm that the isolated bacteria is E Coli. We did oxidase test on the colony obtained from SMAC agar and received oxidase negative result. This was confirmatory to our findings because E Coli is oxidase negative organism. We additionally Gram stained the material and obtained Gram negative bacteria, which also confirmed the result because E. Coli is Gram negative bacteria. In order to further confirm that the bacteria we isolated is E. Coli we used API20E bacterial identification strip and we checked the results using the API web program, and the results were positive for E Coli with 99.6% of accuracy. This result positively identivies E Coli type 0175 to be present in significant amount (1.2 x 104) in the lettuce that we tested. Based on the results we obtained we can conclude that the lettuce was contaminated with fecal bacteria. Salmonella was not identified. This is important because the people that would be presumably infected with this food, if they have salmonella infection we will conclude that the infection is not caused by eating this food. This is because we didn’t find Salmonella spp. in the food sample. However the lettuce tested positive for E. Coli type 0157. This serotype of E. Coli can cause severe gastroenteritis with findings of blood in the stool. In order to fully confirm the diagnosis we will have to make additional microbiological tests of the stool of the children, and if we are able to identify the same bacteria we can confirm that E. Coli type 0157 is the causative agent of this food poisoning outbreak. Nevertheless we can conclude that the food was contaminated with fecal bacteria and there is a high propability that E. Coli is the causative agent. The food that we tested was a fresh lettuce. If we tested a food that was cooked or processed by some other process we will have to take samples from other objects that may be the source of infection, for example the plates that the people eat, water they drink or test the people who prepared the food if they are carriers of some of pathogenic bacteria. Also the bacteria may have been sublethally damaged, but still present in the food. In this case we can inoculate the samples on nonselective agar (tryptase soy agar for example) and incubate it on 37 degrees Celsius for 2 hours in order the bacteria to start the repairing process. Than on top of this layer we can place a selective agar (VRBG for example) that will inhibit all other bacteria except the fecal bacteria. Also we were suspecting on infection rather than intoxication because the late onset of symptoms. The symptoms appeared 3 days after the meal. If it was a case of food poisoning due to intoxication the symptoms should appear much faster (even in the first 6 hours). In our case the children had blood in their stool and this is why we suspect enterohemoragic E. Coli. This bacteria was found in significant amount in the food sample and we can conclude that there is strong indication that E. Coli found on lettuce is the causative agent in this food poisoning outbreak. Read More