Microbial Analysis On Food Commodities Biology Essay

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Standard microbial tests were performed to detect pathogens or spoilage microorganisms in food commodities such as meat, dairy, cereal and poultry. Over a period of 4 days, food samples such as raw foods like meat was analyzed for the presence of Pseudomonas spp., Escherichia coli, Salmonella, Clostridium perfringens and Staphylococcus aureus; while poultry for the presence of Campylobacter jejuni. In addition, finished products like cheese and flour were analyzed for the presence of Listeria monocytogenes and Bacillus cereus respectively. The tests performed followed the Australian/New Zealand Standards for Food Microbiology, AS 5013 series. Reference organisms were run along the commodity samples to provide negative and positive control, while selective media was used to incubate and isolate the different microorganisms. Results in the experiment showed that meat samples tested positive for all the spoilage organisms, except for the anaerobic C. perfringens, which was not present due to prolonged exposure to air during inoculation. For finished food products, L. monocytogenes was present in the cheese sample while B. cereus was present in the flour sample. The microaerophillic C. jejuni was absent in poultry sample, which was unexpected and probably caused by over-exposure of oxygen during inoculation. As such, for more accurate analysis correct handling techniques and speed while inoculating anaerobes must be cultivated.

Introduction:

Microbial spoilage of foods remains a great concern to the food industry as it influences manufacturers, retailers and consumers alike. Growth of pathogens and spoilage microorganisms are unwanted in view of food safety concerns as well as from an economic standpoint. In the meat industry, metabolic activity and growth of spoilage flora represents the main causes of spoilage of meat and meat products, with formation of off-odors, unpleasant flavors, slime and discoloration as clear indicators. Of the most highly populated areas of the animal that contaminate meat are its skin and gastrointestinal tract. These two areas carry a mixed microbial population of micrococci, pseudomonads, staphylococci, yeast and moulds; in addition to contaminants from soil or feces sources. Studies have shown that under refrigerated storage environments, the spoilage would be caused mainly by aerobic Pseudomonas spp. while under anaerobic conditions by various lactic acid bacteria (Muermans at al., 1993). Poor preparation of meat, insufficient heating and exposure to feces would also lead to production of enterotoxins from Clostridium perfringens and Staphylococcus aureus.

Meanwhile, intestinal tract of poultry often contain high numbers of pathogens like Salmonella and Campylobacter, evisceration during butchering processes would often lead to carcass contamination with the gut flora (Adams and Moss, 2008). Camphylobacter jejuni is more pronounced in poultry as carcasses are not separated like those of cattle of sheep, hence effective washing of gut cavities are made more difficult. Further freezing or chilling of poultry meat in water also offers further opportunities for cross contamination.

Amongst the leading cause of death in foodborne bacterial pathogens is Listeriosis, caused by the bacterium Listeria monocytogenes (Ramaswamy et al., 2007). This gram positive, intracellular bacterium has been associated with dairy foods such as raw milk and soft ripened cheese, and is able to grow and multiply in low temperature conditions. Therefore in the microbial analysis of cheese in this experiment, we carried out tests for the detection of this virulent foodborne pathogen. On the other hand, in cereal foods like flour, the most commonly found organisms would be moulds such as Aspergillus sp., Penicillium sp., Fusarium sp. etc. or Bacillus species. The most important environmental factors that influence the growth of spoilage in cereals are water activity and temperature. Gram positive, facultative B. cereus is harmful as it causes minor foodborne illnesses that have diarrheal and emetic symptoms. This problem arises from improper refrigeration of food that allows endospores to germinate and produce enterotoxins, ingestion would then result in nausea, diarrhea and vomiting (Ehling-Schulz et al., 2004).

The objective of this experiment was to conduct microbial analysis of different food commodities, some raw and some finished products used throughout the food industry. Meat, cheese, cereal and poultry samples were examined for presence of pathogenic or spoilage microorganisms, as well as bacteria and fungi.

Materials and methods:

Meat

25g of meat sample and 225mL of buffered peptone water was added together and homogenized in stomacher for few minutes. Meat, raw and unprocessed was then analyzed for unknown pathogenic and spoilage organisms such as Pseudomonas spp., Escherichia coli, Salmonella, Clostridium perfringens and Staphylococcus aureus.

Clostridium perfringens test

Reference organism: Clostridium perfringens (positive)

Sample dilutions of 10-2, 10-3, and 10-4 of food sample were performed in thioglycollate broth, by placing 1mL of sample in 9mL of broth. Next, 0.1mL was pipetted out and spread over surface of Tryptose sulfite cycloserine (TSC) agar, a total of 3 plates. Repeat spread plate method for reference organism, but 0.1mL from broth was taken from the bottom of the anaerobe vial. Total of 4 plates, incubate in anaerobic conditions at 37°C for 24 hours. After 24 hours, examined and counted black colonies, when not present, do not conduct further tests.

Salmonella test

Reference organism: Salmonella ssp. (positive); Escherichia coli (negative)

0.1mL sample was inoculated into Rappaport Vassiliadis (RV) medium and incubated at 42°C for 24 hours, while 1mL of sample was placed into Mannitol selenite cystine (MSC) broth and incubated at 37°C for 24 hours. Sterile, flamed loop was used to scrape a few colonies from reference organisms and inoculated in one MSC broth and one RV broth for each. Total of 3 RV and 3 MSC broths. After 24 hours, turbidity of broth was examined. 6 plates of Xylose lysine desoxycholate (XLD) agar and 6 plates of Bismuth sulfite agar (BSA) were looped with each culture, plates were incubated at 37°C for 24 hours. After 24 hours, examined for typical colonies. Positive colonies were stab inoculated into Sulphite indole motility (SIM) media, stab streaked in Triple sugar ion (TSI) media and streaked on CLED agar. All cultures were incubated at 37°C for 24 hours. After incubation, CLED and TSI were examined; indole test was done on SIM media, observations were recorded down.

Escherichia coli test

Reference organism: Escherichia coli (positive); Enterobacter aerogenes (negative)

1mL of sample was inoculated into triplicate tubes containing 9mL Lauryl Tryptose (LT) broth and incubated at 37°C for 24 hours, another 1mL was placed inside triplicate tubes of EC broth and incubated at 44°C for 24 hours. Reference samples were inoculated into two control tubes each, total of 5 LT broths and 5 EC broths. After 24 hours, checked for presence of gas or bubbles, all presumptive positive cultures were subcultured onto Eosin methylene blue (EMB) agar and incubated at 37°C for 24 hours. After 24 hours, examined for typical colonies. Positive colony was stab inoculated in SIM media at 37°C for 24 hours. After incubation, indole test was done on SIM media, observations were recorded down.

Pseudomonas spp. test

Reference organism: Pseudomonas fluorescens (positive); Pseudomonas aeruginosa (positive)

Sample dilutions of 10-1 (original), 10-2, 10-3 and 10-4 of food sample were performed in 0.1% peptone water, by placing 1mL of sample in 9mL of peptone. Next, in duplicates 0.1mL was pipetted out and spread over surface of Plate count agar (PCA) and on Pseudomonas agar (PA), plates were then sent for incubation at 37°C for 24 hours. Reference organisms were inoculated onto PA and PCA agar by loop and streak method. Total of 7 PA and 7 PCA plates. After 24 hours, checked and counted colonies.

Staphylococcus aureus test

Reference organism: Staphylococcus aureus (positive); Staphylococcus epidermidis (negative)

Sample dilutions of 10-1 (original), 10-2 and 10-3 of food sample were performed in 0.1% peptone water. Next, 0.1mL was pipetted out and spread over surface of Baird-Parker (BP) agar, plate was then sent to incubate at 37°C for 24 hours. Repeat steps for reference organisms, both organisms were streaked onto separate BP plates. After 24 hours, examined and counted black, shiny colonies. Conducted catalase and coagulase test.

Dairy (cheese)

Reference culture: Listeria monocytogenes

25g of cheese and 225mL of BHI broth was added together and homogenized in stomacher for few minutes. As a finished product, this dairy sample was analyzed for the presence of pathogen Listeria monocytogenes.

Homogenized sample was poured into labeled bottle and sent for incubation at 30°C for 24 hours. After 24 hours, streak culture onto Listeria selective agar (LSA). Reference culture was streaked onto separate agar plate. Plates were incubated at 37°C for 24 hours. After another 24 hours, checked for presence of Listeria. Positive colony was streak inoculated onto TSYEA, incubated at 37°C for 24 hours and stab inoculated onto TSYEB, incubated at 25°C for 24 hours. Repeat for reference culture. Conducted catalase test from colony of TYSEA and performed wet mount on colony from TYSEB.

Cereal food (flour)

Reference culture: Bacillus cereus

10g of flour and 90mL of peptone water was added together and homogenized in stomacher for few minutes. As a finished product, this cereal sample was analyzed for moulds or Bacillus species.

Sample dilutions of 10-1 (original), 10-2 and 10-3 of food sample were performed in peptone, by placing 1mL of sample in 9mL of peptone. Next, 0.1mL from all three dilutions was inoculated onto PEMBA and OGYE agar by spread method. Repeat inoculation on PEMBA agar for reference organism by loop and streak method. Total of 4 PEMBA and 3 OGYE plates, incubate PEMBA at 37°C and OGYE at 30°C for 24 hours. After 24 hours, plates were examined and recorded for growth and incubated for a further 24 hours. After another 24 hours, plates were examined again and spore stain was conducted on PEMBA colonies.

Poultry

Reference culture: Campylobacter jejuni

From the selective enrichment broth pre-inoculated and incubated, 0.1mL was inoculated onto Preston agar, Skirrow agar and Blood agar by spread method. Total of 3 plates, incubated at 42°C for 48 hours in microaerobic environment. After 48 hours, examined for typical colonies. Conducted oxidase test for colonies on blood agar, conduct gram stain if results were positive. Conduct wet mount to check for motility.

Results:

Table 1: Observation of Clostridium perfringens presence in meat sample and its positive control.

Clostridium perfringens

Meat sample

Positive control

Clostridium perfringens

Colony apperance on TSC agar. Lecithinase production.

No black colonies, only white colonies observed. Negative result. No lecithinase production.

No black colonies, white colonies observed. Negative result, suspected prolonged exposure to air during inoculation.

Table 2: Observation of Salmonella presence in meat sample and its positive and negative (E.coli) controls.

Salmonella

Meat sample

Positive control

Salmonella ssp

Negative control

Escherichia coli

MSC appearance

Solution turned from transparent to cloudy red.

Solution turned from clear to cloudy and dark red.

Less cloudy, light red solution.

RV appearance

Slight turbidity observed.

Cloudy solution.

Clear solution.

XLD appearance

Red colonies with black centers

Red colonies with black centers.

No growth.

BSA appearance

Few metallic colonies with black centers.

Small metallic colonies with no black center, black precipitate.

No growth.

Sulphite Indole Motility (SIM) medium.

H2S production

Indole test

Motility

Blackening along stab line indicated H2S production.

No color change, indole negative.

Turbidity indication of motile organism.

H2S produced, agar turned black

Indole negative, no color change.

Motile.

Not done as there were no colonies.

Triple Sugar Ion (TSI ) media

Black with red slant, gas production, bubble at stab site.

Black agar with red slant.

Not done as there were no colonies.

CLED agar

Flat blue colonies, yellow colonies indication of contamination.

Pale blue, flat colonies

Not done as there were no colonies.

Table 3: Observation of E.coli presence in meat sample and its positive and negative (E.aerogenes) control.

Escherichia coli

Meat sample

Positive control

Escherichia coli

Negative control

Enterobacter aerogenes

Gas (LT) (37°C)

Positive for gas production as bubble was present.

Positive for gas production as bubble was present, solution was turbid.

No gas production as no bubbles were present, solution was clear.

Gas (EC) (44C)

Positive for gas production due to presence of bubble.

Positive for gas production as bubble was present, solution was cloudy.

No gas produced, clear solution with no bubbles.

EMB (LT) (37°C) appearance

Little green colonies with mettalic sheen and purple centers.

Colonies with green metallic sheen, dark purple center.

-

EMB (EC) (37°C) appearance

Lots of green colonies with metallic sheen, dark purple centers.

Colonies with green metallic sheen, dark purple center.

-

Sulphite Indole Motility (SIM) medium from EC.

Motility

Diffuse growth

Motile, as media had pale layer on top. Diffuse growth as turbidity throughout.

Diffuse growth from stab site, motile. No blackening.

Not done as no colonies were present.

Indole (EC)

Red ring formed, indole positive.

Red ring formed, indole positive.

Red ring formed, indole positive.

Table 4: Observation of Staphylococcus aureus presence in meat sample and its positive and negative (S. epidermidis) control.

Staphylococcus aureus

Meat sample

Positive control

Staphylococcus aureus

Negative control

S. epidermidis

Appearance of BP agar.

Small round, black colonies.

Black colonies observed

Brown colonies observed.

Cell count, cfu/ml

10-1 = 86

10-2 and 10-3 was < 30

-

-

Catalase test

Positive, slight bubbling.

Positive, bubbling occurred.

Positive, bubbling occurred.

Coagulase test

Negative, no cogulation occurred.

Positive, slight coagulation.

Negative, no coagulation.

Table 5: Observation of Pseudomonas presence in meat sample and its positive controls, P. fluorescens and P. aeruginosa.

Pseudomonas

Meat sample

Positive control

P. fluorescens

Positive control

P. aeruginosa

Colony appearance on Pseudomonas agar (PA)

Small round, opaque colonies. No green colonies.

Green colored colonies present.

Light green colonies.

Colony appearance on PCA

Small round, opaque colonies. No green colonies.

Greenish blue colonies observed.

Yellowish green colonies.

Cell count, cfu/ml on PCA

TNTC

-

-

Dairy product: Cheese

Table 6: Observation of Listeria monocytogenes in cheese sample and its positive control.

Listeria monocytogenes

Cheese sample

Positive control

Listeria monocytogenes

Appearance (LSA)

Colonies with dark brown halos.

Clear colorless colonies with dark brown halos.

Apperance on TSYEA

Small opaque round colonies.

Round, white, flat colonies.

Catalase test from TSYEA

Positive, bubbling observed.

Positive, bubbling released.

Appearance of TSYEB

Turbid solution.

Medium was turbid.

Wet mount from TSYEB - motility

Rods observed to be in tumbling movement.

Motile, rod shaped, tumbling movement.

Cereal type: Flour

Table 7: Observation of Bacillus cereus in flour sample and its positive control, Bacillus cereus.

Bacillus cereus

Flour sample

Positive control

Bacillus cereus

OGYE appearance

Big, round opaque circle with black mouldy center.

Large white colonies with black stripes radiating from the center.

PEMBA appearance

Blue colonies with cloudy center. Agar turned from green to completely blue.

Dark grey colonies with fuzzy halo, agar was dark blue.

Spore stain on PEMBA colonies.

Stained green spores, in the middle of rod shaped bacterium.

Spore stained green. Central spores that do not bulge cell were observed.

Table 8: Observation of Bacillus cereus spores that were stained with Schaeffer-Fulton stain.

Moulds

Flour sample

Spore stain (Schaeffer-Fulton stain)

Spore structures

Spores stained green. Observation of central spores that do not bulge the cell.

Identity

Rod shaped bacterium, possible bacillus spores.

Poultry: Campylobacter jejuni

Table 9: Observation of Campylobacter jejuni in poultry sample and its positive control.

Culture

Campylobacter jejuni

Preston agar

No colony growth.

No colony growth.

Skirrow agar

No colony growth.

No colony growth.

Blood agar

No colony growth.

White, translucent colonies with rough, irregular surface.

Oxidase test

-

Negative, no bubbling

Wet mount

Motility

-

Not motile.

Discussion:

The meat sample tested positive for the following microorganisms, of Pseudomonas spp., Escherichia coli, Salmonella and Staphylococcus aureus but negative for Clostridium perfringens. The absence of C. perfringens from both the meat sample and reference cultures was unexpected as the meat sample was raw and left at room temperature for some time. This spore-forming, enterotoxin producing bacterium should have been present in the sample as its vegetative cells makes it fairly heat resistant. C. perfringens is also gram positive, rod shaped and anaerobic, the absence of it on the TSC agar could be explained by prolonged exposure to air during inoculation in the thioglycollate broth, thereby increasing the oxygen levels and creating an unfavorable, aerobic environment.

Both MSC and RV are selective enrichment broth for the isolation of Salmonella, hence the turbidity observed in Table 2 due to the bacterial growth. Further inoculation into BSA yielded positive results of black mettalic colonies surrounded by black percipitate but negative for E.coli. Due to bismuth sulfite and brilliant green that permit the selective growth of Salmonella but inhibits Enterobacteiace (E.coli). Furthermore, XLD indicated the presence of Salmonella as it was able to decarboxylate lysine, cause an alkaline reaction and produce sulphide from the thiosuphate of the medium, the black centered red colonies are a result of sulphide reacting with an iron salt. The presence of flat blue colonies on the CLED medium was characteristic of Salmonella, the medium clearly differentiates the microorganisms found in urine, the presence of yellow colonies in the meat sample indicated contamination from either E.coli or S. aureus. Meanwhile, SIM medium was used to differentiate enteric bacilli based on its sulfide production which turned the agar black, indole negativity and motility. TSI was the indicator medium that contained glucose, lactose, sucrose and ferrous sulfate to categorize Salmonella spp. and other enteric bacteria. H2S production caused the blackening of the medium.

The presence of Escherichia coli was determined through tests such as the presence of gas bubbles in the LT and EC broth, the former had sodium lauryl sulphate as the selective agent for coliform bacteria and Durham tubes detected gas production; while the lactose content of the latter favoured the growth of coliform bacteria that metabolize lactose with gas formation and the bile salts inhibited the growth of gram positive bacteria. Furthermore, SIM medium was used to confirm the enteric organism based on its sulfide production which turned the agar black, indole positivity and motility.

Staphylococcus aureus was detected from the meat sample, as the BP agar exhibited balck colonies with a clear zone of proteolysis, prolonged incubation yielded a more opaque zone. The presence of glycine and pyruvate favored S. aureus growth while the lithium and tellurite inhibited competing bacteria. The reduction of tellurite resulted in balck colonies and the proteolysis of egg protein made a clear zone that surrounded the colonies, further incubation caused an opaque zone due to lipases. Catalase test was positive but the coagualse test was inconclusive, probably due to other bacterial growth on the medium.

The presence of Pseudomonas spp. was confirmed with the presence of growth on the Pseudomonas Agar, as any growth on the media would indicate the species, and the green colonies further indicated presumptive Pseudomonas aeruginose growth. Plate count agar (PCA) however was used to enumerate the total viable cell count, the colonies were over 300 and too numerous to count.

For the dairy product, Listeria monocytogenes was detected in the cheese sample. In LSA, aesculin was added as a differential indicator, hence it gets hydrolysed and reacts with the iron salt to give a black precipitate around the colonies. Both TYSEA and TYSEB contributed to the confirmation of L. monocytogenes. Positive catalase test and the characteristic tumbling motility of the bacterium were observed. In addition to its gram positive, facultative anaerobic and non-sporing atributes. Listeriosis is one of the leading causes of death in foodborne illnesess, hence accurate testing and prevention is needed as it remains one of the most dangerous hazards in the doof industry.

Bacillus cereus was present in the flour sample, a finished cereal product. Gram positive, facultative B. cereus is harmful as it causes minor foodborne illnesses that have diarrheal and emetic symptoms. Mold growth was obserbed as yeast extract stimulated the growth of molds in OGYE Agar Base, with dextrose as the carbon energy source while oxytetracycline inhibited bacterial growth. The PEMBA medium turned from green to blue due to bromothymol blue that acted as a pH indicator, B. cereus can be determined in the medium due to precipitation from lecithin hydrolysis. The vegetative cells in B. cereus imparted heat resistance to the miroorganism, hence when cooked food is refrigerated improperly, the endospores would germinate and result in the production of enterotoxins, highly heat resistant and pH resistant.

In the poultry sample Campylobacter jejuni was absent from both the reference and sample. The result was unexpected as Campylobacter is quite prevalent in poultry, due to cross contamination from highly colonized areas in the animal's gastrointestinal tract. This was probably due to incorrect inoculation techniques such as prolonged exposure of culture to the air, which increased oxygen levels. As the rod shaped, non sporing bacterium is microaerophilic in nature, in needs an environment which containins lower concentration of oxygen than that of the atmosphere. This bacterium represents one of the most common causes of human gastroenteritis.

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