Food Microbiology: Laboratory Report
Pathogenic bacteria such as Salmonella, Clostridium, Listeria and Campylobacter have been implicated in several food poisoning conditions. These bacteria have also been isolated from several processed foods. Research has shown that the bacteria contaminate food and cause deadly foodborne infections to humans. However, few studies have examined the relationship between the freshness of food and its storage and the extent of pathogen contamination. The aim of this experiment was to examine five turkey mince food samples for the total number of bacterial colony forming units and for the presence of these four pathogens by culture method. The five samples were within the sell-by date (sample 1), at sell-by date (sample 2), three days beyond sell-by date (sample 3), one week beyond sell-by date (sample 4) and two weeks beyond sell-by date (sample 5). 2.0 x 103 colony forming units (CFUs) were isolated from sample 1, 2.0 x 104CFUs in sample 2, 7.1 x 104 CFUs in sample 3.42 x 106 CFUs in sample 4 and 2.41 x 107CFUs in sample 5. However, the number of the pathogens in the food samples was not related to the sell-by date of the turkey mince as some pathogens were higher in sample 1 than in sample 5. In conclusion, the amount of bacterial colonies are highest in foods that are obove the expiry date but lowest in those that are within the expiry dates. However, the presence of the pathogenic bacteria does not necessarily depend on the expiry date labels on the food.
This experiment was designed to examine food samples for the presence of pathogens that cause food poisoning using conventional bacterial culture methods. According to Newell et al. (2010), some of the mostly implicated pathogens in food poisoning include Salmonella, Clostridium, Listeria and Campylobacter. Salmonella species is a bacterium that colonise different hosts ranging frommajorlivestock to humans. Therefore, food and food products can easily get contaminated with the bacteria from the different hosts. In addition, the bacteria have the ability to grow in unprocessed food and has a long-term survival rate in favourable environments (Newell et al., 2010). Consequently, food-borne salmonellosis outbreaks have been routinely observed and reported in the United Kingdom. Data from the Public Health England (2014) indicate that different Salmonella serotypes have accounted for food poisoning, typhoid fever and paratyphoid fever in more than 14,000 reported cases in England. The food poisoning due to Salmonella species contamination is characterised by diarrhoea, nausea, stomach cramps and sometimes fever may develop between 12 and 72 hours after infection. These symptoms usually resolve within 5 to 7 days and therefore do not require any treatmentmethods other than oral fluids and rehydration agents to replenish the lost fluids and minerals. However, severe cases of the disease are treated with antibiotics following a culture and sensitivity test(Reis & Horn, 2010).
Clostridium difficultand C. perfrigens areother bacterial species that has been implicated in food poisoning. Rodriguez-Palacios, Staempfli, Duffield, & Weese ( 2007)reports a successful isolation of the bacteria in 20% of all the sampled retail meat in Canada. Gould & Limbago(2010) also confirms their hypotheses that the bacteria is responsible for the transmission of several foodborne infections to humans. The bacteria produceClostridium perfrigens toxin which is responsible for thetype A food poisoning(Brynestad & Granum, 2002). The toxin targets epithelial cells where they interfere with the plasma membrane permeability. This leads to symptoms such as abdominal cramps and watery diarrhea that usually appear between 6 and 24 hours following ingestion of the contaminated food (Brynestad & Granum, 2002). The therapy for the poisoning involves the management of the complications until the disease ends. The management involves the intake of rehydrating fluids, eating healthy diets and replacing the lost salts. Sometimes, antibiotics can be used when the symptoms persist.
Listeria monocytogenes have also been implicated in several food poisoning conditions characterised by a high mortality rate of between 20 and 30% (Newell et al., 2010). Listeriosis is, therefore, a fatal invasive disease which is majorly reported in neonates, pregnant women, the elderly and the immunocompromised(Swaminathan & Gerner-Smidt, 2007). The bacteria are resistant to refrigeration temperatures whichincrease its survival in foods. Upon ingestion, the bacteria are internalised by the host’s gastrointestinal epithelial cells where it undergoes multiplication and initiates the infection process (Poulsen & Czuprynski, 2013). During the internalisation, the bacteria produce a toxin called thelisteriolysinwhich lyses the vacuole of the cell allowing its entry into the host cell cytoplasm. The bacteria also have the ability to cross the blood-brain barrier and the fetoplacental barrier and thus causes brain and neonatal infections. The infections are present clinically in two forms; the noninvasive and the invasive forms. Most cases of the infection are non-invasive characterised by flu-like symptoms such as fever and arthromyalgia. In addition, the noninvasive form is symptomed by Listeria gastroenteritis involving non-bloody watery diarrhea, nausea, vomiting and abdominal cramps(Bortolussi, 2008).
The invasive form of the disease is the most dangerous type often associated with high infant mortality. When there is an infection in the brain, the infection presents in the form of meningitis often characterised by confusion, convulsions, headache and nuchal rigidity. The signs are more pronounced in neonates and children than in adults(Lamont et al., 2011). Immunocompromised adults also have severe symptoms of listeria meningitis. The infection in children occurs after several hours to days after birth following acquisition of the bacteria from the mother during pregnancy(Lamont et al., 2011). The invasive form of the disease also leads to respiratory complications, jaundice, fever, rash and lethargy (Bortolussi, 2008).
The treatment of listeriosis depends on the stage of the infection(Allerberger & Wagner, 2010). The non-invasive form of the disease rarely requires antimicrobial therapy because the symptoms often resolve within two days. The progression of the disease to the invasive form is uncommon except in the immunocompromised, the pregnant and the elderly. However, if the disease progresses to the severe form, treatment is vitally important. The treatment involves antimicrobial chemotherapy using penicillin and ampicillin. Additional therapy is also necessary to prevent the bacteria from reaching the central nervoussystem(Allerberger & Wagner, 2010).
Lastly, Campylobacter species have also been associated with several food poisoning conditions. According to Newell et al. (2010), Campylobacterspecies is the second most common cause of bacterial food poisoning after Salmonella in the world. In the United Kingdom, Inns, Foster, & Gorton (2010) finds that 13 cases out of 24 cases of gastroenteritis among guests at a wedding reception were due toCampylobacter infection. According to Janssen et al. (2008), Campylobacter induces disease in humans by two mechanisms. First, the bacteria adhere to the intestines and produces toxins which alter the resorption capacity of the intestines. This leads to secretory diarrhea (Janssen et al., 2008). Secondly, the bacteria can invade the intestinal mucosa, multiply and cause inflammatory responses. Consequently, the invasive form of Campylobacter infection is characterised by blood-tinged inflammatory diarrhea (Janssen et al., 2008). The disease is usually restricted to the intestines with symptoms such as diarrhea, nausea and abdominal pains. However, research has shown that in some cases the bacteria can pass through the intestinal mucosa to the extra-intestinal sites through the lymphatic vessels. This leads to systemic infection accompanied by headache, multi-organ infections and inflammatory responses(Zilbauer, Dorrell, Wren, & Bajaj-Elliott, 2008). Campylobacter infection can be treated depending on the level of infection. The intestinal stage ofCampylobacter infectionis managed by the replacement of the lost fluids and electrolytes(Kirkpatrick & Tribble, 2011). The patients are rehydrated with oral glucose-electrolyte drink solutions. The invasive form of the disease requires the use of antibiotics such as erythromycin. Individuals with low haemoglobin levels due to bacteremia requires the transfusion of blood with antibiotics (Kirkpatrick & Tribble, 2011).
All these bacteria cause food spoilage and can be detected by the general characteristics of spoiled food. These include strange odor, discoloration, formation of biofilms and other abnormalobservable characteristics(Bai & Rai, 2011). However, in some cases the food may lack the signs although they contain the contaminating microorganisms.Oftenly, fresh foods well within the sell-by dates are expected to containlittle-contaminatingorganismsthan those that are beyond the sell-by date. Therefore,date labels on food can be used as a guideline to determine the quality of food but due to poor storage environment, the food can become contaminated or spoiled even before the expiry date. Consequently, there is need to assess the quality of the food sold in the market and those consumed. The analysis of the quality of food involves the selection of food sample, culturing the samples and detecting the presence of the contaminating pathogenic bacteria in them. In this experiment, a food sample was examined for the presence of four pathogens; Salmonella, Clostridium, Listeria and Campylobacter. This was done by using 5 samples of turkey mince with variations in sell-by dates. These samples were prepared, serially diluted and inoculated onto plate count agar (PCA) plates for total colony count. The specific pathogens in the samples were then determined using different techniques. It is hypothesised that turkey mince food product with an age that exceeds the sell-by date are more contaminated with these pathogens than those within the sell-by date.
Materials and Method
The following materials were used in the experiment.
- Sample 1 with an age well within the sell-by date
- Sample 2 at sell-by date age
- Sample 3 at three days beyond the sell-by date
- Sample 4 at one week beyond the sell-by date
- Sample 5 at two weeks beyond the sell-by date
- Reagents and Culture media
– Peptone water, plate count agar (PCA) plates, molten oleandomycin polymixin sulphadiazine perfringens (OPSP) agar, Rappaport-Vassiliadis (RV) broth, xylose lysine deoxycholate (XLD) agar, Gram stain reagents, Listeria Enrichment Broth (LEB), Listeria selective agar (LSA) and Campylobacter blood-free selective agar containing CCDA selective supplement
- Other materials
– Universal bottles, weighing machine, spreader, refrigerators, incubators, petri dish, OPSP medium, inoculation loops,a stomacher, microscope and a source of heat.
The following methods was used during the experiment.
- Preparation of the Food Samples
10 grams was aliquoted from each food sample and placed in 9 ml of peptone water. The mixture was homogenised in a stomacher and then transferred to five different sterile universal tubes.
- Total bacterial count
The prepared food suspensions were serially diluted using 1 ml + 9 ml peptone water to obtain 10-2, 10-3, 10-4 and 10-5 dilutions. An aliquot of 0.1 ml from each dilution was inoculated onto plate count agar (PCA) plates and spread using a sterile spreader. The culture plates were incubated at 37oC for 24 hours and then refrigerated. The plates were then examined at a later date and the number of the colonies counted and recorded.
- Detection of Clostridium perfringens
The undiluted mince suspensions were mixed with 9 ml of oleandomycin polymixin sulphadiazine perfringens (OPSP) agar. The mixture was then poured into a sterile petri dish and allowed to solidify. A further 9 ml of the OPSP medium was added to the petri dish to overlay it. The plates were incubated at 37oC for 24 hours and refrigerated. Colonies that developed black halos were counted and recorded. A Gram stain was also performed on one of the putative Clostridium perfringens colonies.
- Detection of Salmonella enterica
After the detection of Clostridium perfringensin the previous method, the remaining homogenate was incubated at 30oC for 72 hours to provide an enrichment for the recovery of the injured organisms. This increases the number of recoverable organisms for the remaining procedures. 1 ml of the pre-enrichment culture was transferred into a tube containing 9 ml of Rappaport-Vassiliadis (RV) broth. The broth was incubated at 41.5oC for 24 hours. After the incubation, the RV broth was subcultured by streaking onto xylose lysine deoxycholate (XLD) agar to give single colonies. The XLD plates were then incubated at 37oC for 24 hours and refrigerated. Later on, the plates were observed for pink colonies with black centre. These colonies were counted, and a gram stain for the putative salmonellae colonies performed.
- Detection of Listeria species
The remaining homogenate was incubated at 30oC for 72 hours to provide an enrichment that increases the number of recoverable organisms. 1 ml of the pre-enrichment culture was transferred into a tube containing 9 ml of Listeria Enrichment Broth (LEB). The broth was then incubated at 30oC for 24 hours. The LEB broth was then subcultured by streaking on Listeria selective agar (LSA) to give single colonies. The agar LSA agar was incubated at 30oC for 48 hours and refrigerated. The plates were observed for black or grey colonies. The colonies were counted and recorded. A gram stain was also performed on the putative Listeria species colonies
- Detection of Campylobacter jejuni
An aliquot of 50 µl of the mince homogenate was added to Campylobacter blood-free selective agar containing CCDA selective supplement. The liquid was then streaked using a sterile wire loop and incubated at 42oC for 48 hours under anaerobic conditions. The plates were then observed for putative C. jejuni colonies using the demonstration colonies. A Gram stain was also performed on one of the putative Campylobacter jejunicolonies
Table one below gives the results of the total count for the different dilutions of the homogenate samples.
|Turkey mince sample number|
|10-2||22, 37, 5, 1, 18, 23, 34||69, 52, 46, 93, 153||270, 280|
|10-3||12, 8||97, 50, 71, 65|
|10-4||163, 100, 72, 172, 104, 173|
|10-5||70, 67, 18, 9, 12||173, 198, 240, 117, 356, 261, 268, 207, 338, 250|
Table 1: Raw data for the total colony count for the different food samples
Table 2 below gives the mean of the total colony count for the five turkey mince food samples. In sample 1, the 10-1 dilution provided 11 colonies and the 10-2 provided an average of 20 colonies (standard deviation =12.47). In sample 2, the dilution 10-2 produced an average of 83 colonies (standard deviation = 43.37) while dilution 10-3 produced a mean of 20 colonies (standard deviation = 4.24). In sample 3 a mean of 275 (standard deviation = 7.07) colonies were obtained in the 10-2 dilution and a mean of 71 (standard deviation = 19.60)colonies in dilution 10-3. In sample 4, dilutions 10-1 to 10-3 had uncountable colonies. However, dilution 10-4 produced an average of 131 colonies (standard deviation = 43.91) while dilution 10-5 produced a mean of 42 colonies (standard deviation = 31.00). Sample 5 also produced uncountable number of colonies in dilutions 10-1 to 10-4. However, dilution 10-5 produced a mean of 241 colonies (standard deviation = 72.15).These results indicate that all the food samples were contaminated with bacteria.
|Turkey mince sample number|
Table 2: Mean total colony count for the different food samples.
The number of colonies in the four food samples were calculated as follows
Sample 1 = 20 x 102
= 2.0 x 103 CFUs
Sample 2 = 20 x 103
= 2.o x 104 CFUs
Sample 3 = 71 x 103
= 7.1 x 104 CFUs
Sample 4 = 42 x 105
= 4.2 x 106 CFUs
Sample 5 = 241 x 105
= 2.41 x 107 CFUs
From the above results, sample 5 had the highest number of colonies while sample 1 had the lowest number of colonies.
Table 3 shows the results obtained for the presence or absence of the specific pathogens in the five food samples. The number of Salmonella species was highest in sample 5 where9 colonies of the bacteria was counted. The number was lowest in sample 3 where only 7 colonies of the bacteria was counted. Sample 5 also recorded the highest number of Clostridium spp contamination having 9 colonies. However, the sample had the lowest number of Listeria spp.
|Salmonella spp.||Clostridium spp.||Listeria spp.||Campylobacter spp.|
Table 3: Qualitative data (presence/absence) of potentially pathogenic bacterial genera in the five food samples
Table 4 shows the Gram stain results for the putative Salmonellae sppcolonies, putativeClostridium perfringens, putative Listeria species colonies and putative Campylobacter jejunicolonies. Gram staining revealed that the putative Salmonellae colonies were Gram negative and rod-shaped. The putative Clostridium perfringens colonies were Gram positive and also rod-shaped. The putative Listeria species colonies appeared as Gram-positive rods. The colonies of Campylobacter jejuni were Gram negative and helical in shape. These results confirmed the Gram stain characteristics and morphology of the bacteria.
|Clostridium perfringens||Purple||Gram-positive rods|
|Salmonellae spp||Red||Rods||Gram-negative rods|
|Listeria species||Purple||Rods||Gram-positive rods|
Table 4: Gram stain results for the isolated colonies
The results of the study confirm the hypotheses that food products with an age that exceeds the sell-by date are more contaminated with bacterial pathogens than those with an age within the sell-by date. For instance, Sample 5 which had an age of two weeks beyond the sell-by date had the highest number of bacterial colonies (2.41 x 107 colony forming units). Sample 1 which was well within the sell-by date had the lowest number of colonies (2.0 x 104colony forming units). However, the presence of specific pathogens in the food did not follow the sell-by date criteria. Although sample 5 recorded the highest number of Salmonella spp, Clostridium spp and Campylobacter spp, it did not contain the highest number of Listeria spp. Unexpectedly, Listeria spp.was highest in sample 1 which was well within the sell-by date and was expected to contain few of any such pathogens.
In addition, sample 2, which was at the sell-by date, had the same number of Salmonella spp. colonies as sample 5.A possible explanation for this discrepancy may be post-processing contamination with the specific pathogen or poor storage conditions considering that Listeria spphas the ability to multiply under refrigeration temperatures. Thesediscrepancies makes it difficult to conclude that the number of pathogens in minced turkey increases as the sell-by date increases. Perhaps the only trend followed by the number of contaminating pathogens in meat is the processing conditions, storage conditions and possible post-processing contamination such as unhygienic handling of processed food. Despite these differences, the study successfully demonstrates the presence of four pathogens in the food samples. There was no sample without any of the four pathogens. Therefore, it is deductible that all the minced turkey meat had four pathogenic bacteria; Salmonella spp, Clostridium spp, Listeria sppand Campylobacter spp.
A study by Oranusi, Braide, & Osigwe (2012) also confirms that the expiry date of food does not necessarily influence the type and number of pathogenic microorganisms isolated from the food samples. In a study that involved only food samples that were within the expiry dates, Oranusi et al. (2012) isolated Staphylococcus aureus, Salmonella spp. Bacillus subtilis, Clostridium perfringens and other food-poisoning pathogens in the canned food samples. This study provides enough evidence to prove that there is a possibility of microbial contamination of foods that are still within the expiry dates.
The results of this experiment are coherent with several other studies. For instance, a study by Remm, Koch, Von Muffling & Nowak (2009) demonstrated the presence of two of these pathogens and Pseudomonas spp in unseasoned minced turkey meat. However, the study did not find Campylobacter sppand Clostridium sppin the samples. The results of the study showed that Listeria monocytogenes were the most frequent pathogen in the samples, present in 7 of all the 48 samples. Salmonella spp was found to be present in only one of the samples(Remm et al., 2009). This study confirms the presence of Salmonella and Listeria spp in turkey mince. Although this study does not specify the sell-by dates of the food samples, the researcher demonstrated the presence of these pathogens in the food. This research is, therefore, in coherence with the results of this experiment.
Tassew, Abdissa, Beyene & Gebre-Selassie (2010) also analysed the microbial quality of minced meat from different butcheries and hotels. The study finds that there were different pathogenic microorganisms and indicator organisms in the food samples. For example, microbiological analysis of the 65 samples revealed that 2 samples (1.2%) had Salmonella spp bacteria(Tassew et al., 2010). This study also found other bacteria such as Pseudomonas,Citrobacter, Providencia, Escherichia coli and other bacteria of the Enterobacteriaceae family contaminating the food samples(Tassew et al., 2010). Although this study did not exclusively analyse minced turkey or minced meat, it demonstrated the presence of pathogenic bacteria in food.
This study demonstrates the presence of four significant food poisoning pathogenic bacteria in turkey mince. Thes pathogens include Salmonella spp, Clostridium spp, Listeria sppand Campylobacter spp. The presence of the pathogenic bacteria does not necessarily depend on the expiry date labels on the food. However, food with an age of one or more weeks beyond the sell-by date contain higher bacterial colony forming units compared to those that are within the sell-by date. To some extent, the amount of these bacterial pathogens is also highest in food that are above the expiry dates. The bacterial pathogens contaminating foodstuffs causes deadly human diseases some involving the brain and other vital body organs. Therefore, there is a need for an effective program that will prevent under-processing, pre-processing and post-processing contamination of food.
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