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Standard microbic trials were performed to observe pathogens or spoilage micro-organisms in nutrient trade goods such as meat, dairy, cereal and domestic fowl. Over a period of 4 yearss, nutrient samples such as natural nutrients like meat was analyzed for the presence of Pseudomonas spp. , Escherichia coli, Salmonella, Clostridium perfringens and Staphylococcus aureus ; while domestic fowl for the presence of Campylobacter jejuni. In add-on, finished merchandises like cheese and flour were analyzed for the presence of Listeria monocytogenes and Bacillus cereus severally. The trials performed followed the Australian/New Zealand Standards for Food Microbiology, AS 5013 series. Reference beings were run along the trade good samples to supply negative and positive control, while selective media was used to incubate and insulate the different micro-organisms. Consequences in the experiment showed that meat samples tested positive for all the spoilage beings, except for the anaerobiotic C. perfringens, which was non present due to drawn-out exposure to air during vaccination. For finished nutrient merchandises, L. monocytogenes was present in the cheese sample while B. Cereus was present in the flour sample. The microaerophillic C. jejuni was absent in domestic fowl sample, which was unexpected and likely caused by over-exposure of O during vaccination. As such, for more accurate analysis correct managing techniques and velocity while inoculating anaerobes must be cultivated.

Introduction:

Microbial spoilage of nutrients remains a great concern to the nutrient industry as it influences makers, retail merchants and consumers likewise. Growth of pathogens and spoilage micro-organisms are unwanted in position of nutrient safety concerns every bit good as from an economic point of view. In the meat industry, metabolic activity and growing of spoilage vegetation represents the chief causes of spoilage of meat and meat merchandises, with formation of off-odors, unpleasant spirits, sludge and stain as clear indexs. Of the most extremely populated countries of the animate being that contaminate meat are its tegument and GI piece of land. These two countries carry a assorted microbic population of Micrococcus, pseudomonads, staphylococcus, barm and molds ; in add-on to contaminations from dirt or fecal matters beginnings. Surveies have shown that under refrigerated storage environments, the spoilage would be caused chiefly by aerophilic Pseudomonas spp. while under anaerobiotic conditions by assorted lactic acid bacteriums ( Muermans at al. , 1993 ) . Poor readying of meat, deficient warming and exposure to fecal matters would besides take to production of enterotoxins from Clostridium perfringens and Staphylococcus aureus.

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Meanwhile, enteric piece of land of domestic fowl frequently contain high Numberss of pathogens like Salmonella and Campylobacter, evisceration during butchery procedures would frequently take to carcass taint with the intestine vegetation ( Adams and Moss, 2008 ) . Camphylobacter jejuni is more marked in domestic fowl as carcases are non separated like those of cowss of sheep, therefore effectual lavation of intestine pits are made more hard. Further freeze or cooling of domestic fowl meat in H2O besides offers farther chances for cross taint.

Amongst the taking cause of decease in foodborne bacterial pathogens is Listeriosis, caused by the bacteria Listeria monocytogenes ( Ramaswamy et al. , 2007 ) . This gm positive, intracellular bacteria has been associated with dairy nutrients such as natural milk and soft ripened cheese, and is able to turn and multiply in low temperature conditions. Therefore in the microbic analysis of cheese in this experiment, we carried out trials for the sensing of this deadly foodborne pathogen. On the other manus, in cereal nutrients like flour, the most normally found beings would be molds such as Aspergillus sp. , Penicillium sp. , Fusarium sp. etc. or Bacillus species. The most of import environmental factors that influence the growing of spoilage in cereals are H2O activity and temperature. Gram positive, facultative B. Cereus is harmful as it causes minor foodborne unwellnesss that have diarrheal and emetic symptoms. This job arises from improper infrigidation of nutrient that allows endospores to shoot and bring forth enterotoxins, consumption would so ensue in sickness, diarrhoea and emesis ( Ehling-Schulz et al. , 2004 ) .

The aim of this experiment was to carry on microbic analysis of different nutrient trade goods, some altogether and some finished merchandises used throughout the nutrient industry. Meat, cheese, cereal and domestic fowl samples were examined for presence of pathogenic or spoilage micro-organisms, every bit good as bacteriums and Fungis.

Materials and methods:

Meat

25g of meat sample and 225mL of buffered peptone H2O was added together and homogenized in stomacher for few proceedingss. Meat, natural and unrefined was so analyzed for unknown pathogenic and spoilage beings such as Pseudomonas spp. , Escherichia coli, Salmonella, Clostridium perfringens and Staphylococcus aureus.

Clostridium perfringens trial

Mention being: Clostridium perfringens ( positive )

Sample dilutions of 10-2, 10-3, and 10-4 of nutrient sample were performed in thioglycollate stock, by puting 1mL of sample in 9mL of stock. Next, 0.1mL was pipetted out and spread over surface of Tryptose sulfite cycloserine ( TSC ) agar, a sum of 3 home bases. Repeat dispersed home base method for mention being, but 0.1mL from stock was taken from the underside of the anaerobe phial. Sum of 4 home bases, incubate in anaerobiotic conditions at 37 & A ; deg ; C for 24 hours. After 24 hours, examined and counted black settlements, when non present, do non carry on farther trials.

Salmonella trial

Mention being: Salmonella ssp. ( positive ) ; Escherichia coli ( negative )

0.1mL sample was inoculated into Rappaport Vassiliadis ( RV ) medium and incubated at 42 & A ; deg ; C for 24 hours, while 1mL of sample was placed into Mannitol selenite cystine ( MSC ) stock and incubated at 37 & A ; deg ; C for 24 hours. Sterile, flamed cringle was used to grate a few settlements from mention beings and inoculated in one MSC stock and one RV stock for each. Sum of 3 RV and 3 MSC stocks. After 24 hours, turbidness of stock was examined. 6 home bases of Xylose lysine desoxycholate ( XLD ) agar and 6 home bases of Bismuth sulfite agar ( BSA ) were looped with each civilization, home bases were incubated at 37 & A ; deg ; C for 24 hours. After 24 hours, examined for typical settlements. Positive settlements were stab inoculated into Sulphite indole motility ( SIM ) media, pang streaked in Triple sugar ion ( TSI ) media and streaked on CLED agar. All civilizations were incubated at 37 & A ; deg ; C for 24 hours. After incubation, CLED and TSI were examined ; indole trial was done on SIM media, observations were recorded down.

Escherichia coli trial

Mention being: Escherichia coli ( positive ) ; Enterobacter aerogenes ( negative )

1mL of sample was inoculated into triplicate tubings incorporating 9mL Lauryl Tryptose ( LT ) stock and incubated at 37 & A ; deg ; C for 24 hours, another 1mL was placed inside triplicate tubings of EC stock and incubated at 44 & A ; deg ; C for 24 hours. Mention samples were inoculated into two control tubings each, sum of 5 LT stocks and 5 EC stocks. After 24 hours, checked for presence of gas or bubbles, all presumptive positive civilizations were subcultured onto Eosin methylene blue ( EMB ) agar and incubated at 37 & A ; deg ; C for 24 hours. After 24 hours, examined for typical settlements. Positive settlement was stab inoculated in SIM media at 37 & A ; deg ; C for 24 hours. After incubation, indole trial was done on SIM media, observations were recorded down.

Pseudomonas spp. trial

Mention being: Pseudomonas fluorescens ( positive ) ; Pseudomonas aeruginosa ( positive )

Sample dilutions of 10-1 ( original ) , 10-2, 10-3 and 10-4 of nutrient sample were performed in 0.1 % peptone H2O, by puting 1mL of sample in 9mL of peptone. Following, in extras 0.1mL was pipetted out and spread over surface of Plate count agar ( PCA ) and on Pseudomonas agar ( PA ) , home bases were so sent for incubation at 37 & A ; deg ; C for 24 hours. Reference beings were inoculated onto PA and PCA agar by cringle and run method. Total of 7 PA and 7 PCA home bases. After 24 hours, checked and counted settlements.

Staphylococcus aureus trial

Mention being: Staphylococcus aureus ( positive ) ; Staphylococcus epidermidis ( negative )

Sample dilutions of 10-1 ( original ) , 10-2 and 10-3 of nutrient sample were performed in 0.1 % peptone H2O. Next, 0.1mL was pipetted out and spread over surface of Baird-Parker ( BP ) agar, home base was so sent to incubate at 37 & A ; deg ; C for 24 hours. Repeat stairss for mention beings, both beings were streaked onto separate BP home bases. After 24 hours, examined and counted black, glistening settlements. Conducted catalase and coagulase trial.

Dairy ( cheese )

Reference civilization: Listeria monocytogenes

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

Homogenized sample was poured into labelled bottle and sent for incubation at 30 & A ; deg ; C for 24 hours. After 24 hours, streak civilization onto Listeria selective agar ( LSA ) . Reference civilization was streaked onto separate agar home base. Home plates were incubated at 37 & A ; deg ; C for 24 hours. After another 24 hours, checked for presence of Listeria. Positive settlement was streak inoculated onto TSYEA, incubated at 37 & A ; deg ; C for 24 hours and pang inoculated onto TSYEB, incubated at 25 & A ; deg ; C for 24 hours. Repeat for mention civilization. Conducted catalase trial from settlement of TYSEA and performed wet saddle horse on settlement from TYSEB.

Cereal nutrient ( flour )

Reference civilization: Bacillus cereus

10g of flour and 90mL of peptone H2O was added together and homogenized in stomacher for few proceedingss. As a finished merchandise, this cereal sample was analyzed for molds or Bacillus species.

Sample dilutions of 10-1 ( original ) , 10-2 and 10-3 of nutrient sample were performed in peptone, by puting 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 vaccination on PEMBA agar for mention being by cringle and run method. Total of 4 PEMBA and 3 OGYE home bases, incubate PEMBA at 37 & A ; deg ; C and OGYE at 30 & A ; deg ; C for 24 hours. After 24 hours, home bases were examined and recorded for growing and incubated for a farther 24 hours. After another 24 hours, home bases were examined once more and spore discoloration was conducted on PEMBA settlements.

Domestic fowl

Reference civilization: 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 home bases, incubated at 42 & A ; deg ; C for 48 hours in microaerobic environment. After 48 hours, examined for typical settlements. Conducted oxidase trial for settlements on blood agar, conduct gm discoloration if consequences were positive. Conduct wet saddle horse to look into for motility.

Consequences:

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 settlements, merely white settlements observed. Negative consequence. No lecithinase production.

No black settlements, white settlements observed. Negative consequence, suspected drawn-out exposure to air during vaccination.

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 visual aspect

Solution turned from transparent to cloudy ruddy.

Solution turned from clear to cloudy and dark ruddy.

Less cloudy, light ruddy solution.

RV visual aspect

Slight turbidness observed.

Cloudy solution.

Clear solution.

XLD visual aspect

Red settlements with black centres

Red settlements with black centres.

No growing.

BSA visual aspect

Few metallic settlements with black centres.

Small metallic settlements with no black centre, black precipitate.

No growing.

Sulphite Indole Motility ( SIM ) medium.

H2S production

Indole trial

Motility

Blackening along stab line indicated H2S production.

No colour alteration, indole negative.

Turbidity indicant of motile being.

H2S produced, agar turned black

Indole negative, no colour alteration.

Motile.

Not done as there were no settlements.

Ternary Sugar Ion ( TSI ) media

Black with ruddy angle, gas production, bubble at stab site.

Black agar with ruddy angle.

Not done as there were no settlements.

CLED agar

Flat blue settlements, xanthous settlements indicant of taint.

Pale blue, level settlements

Not done as there were no settlements.

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 & A ; deg ; 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 & A ; deg ; C ) visual aspect

Small green settlements with mettalic shininess and purple centres.

Colonies with green metallic shininess, dark purple centre.

EMB ( EC ) ( 37 & A ; deg ; C ) visual aspect

Tonss of green settlements with metallic shininess, dark purple centres.

Colonies with green metallic shininess, dark purple centre.

Sulphite Indole Motility ( SIM ) medium from EC.

Motility

Diffuse growing

Motile, as media had pale bed on top. Diffuse growing as turbidness throughout.

Diffuse growing from stab site, motile. No darkening.

Not done as no settlements 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 unit of ammunition, black settlements.

Black settlements observed

Brown settlements observed.

Cell count, cfu/ml

10-1 = 86

10-2 and 10-3 was & A ; lt ; 30

Catalase trial

Positive, slight bubbling.

Positive, bubbling occurred.

Positive, bubbling occurred.

Coagulase trial

Negative, no cogulation occurred.

Positive, little curdling.

Negative, no curdling.

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 visual aspect on Pseudomonas agar ( PA )

Small unit of ammunition, opaque settlements. No green settlements.

Green colored settlements present.

Light green settlements.

Colony visual aspect on PCA

Small unit of ammunition, opaque settlements. No green settlements.

Green blue settlements observed.

Yellow green settlements.

Cell count, cfu/ml on PCA

TNTC

Dairy merchandise: 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 aura.

Clear colorless settlements with dark brown aura.

Apperance on TSYEA

Small opaque unit of ammunition settlements.

Round, white, level settlements.

Catalase trial from TSYEA

Positive, bubbling observed.

Positive, bubbling released.

Appearance of TSYEB

Turbid solution.

Medium was turbid.

Wet saddle horse from TSYEB – motility

Rods observed to be in toppling motion.

Motile, rod shaped, toppling motion.

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 visual aspect

Big, unit of ammunition opaque circle with black mouldy centre.

Large white settlements with black chevrons radiating from the centre.

PEMBA visual aspect

Blue settlements with cloudy centre. Agar turned from green to wholly blue.

Dark Grey settlements with fuzzed aura, agar was dark blue.

Spore discoloration on PEMBA settlements.

Stained green spores, in the center of rod shaped bacteria.

Spore stained green. Cardinal spores that do non pouch cell were observed.

Table 8: Observation of Bacillus Cereus spores that were stained with Schaeffer-Fulton discoloration.

Molds

Flour sample

Spore discoloration ( Schaeffer-Fulton discoloration )

Spore constructions

Spores stained green. Observation of cardinal spores that do non pouch the cell.

Identity

Rod shaped bacteria, possible B spores.

Domestic fowl: Campylobacter jejuni

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

Culture

Campylobacter jejuni

Preston agar

No settlement growing.

No settlement growing.

Skirrow agar

No settlement growing.

No settlement growing.

Blood agar

No settlement growing.

White, translucent settlements with unsmooth, irregular surface.

Oxidase trial

Negative, no bubbling

Wet saddle horse

Motility

Not motile.

Discussion:

The meat sample tested positive for the undermentioned micro-organism, 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 mention civilizations was unexpected as the meat sample was natural and left at room temperature for some clip. This spore-forming, enterotoxin bring forthing bacteria should hold been present in the sample as its vegetive cells makes it reasonably heat immune. C. perfringens is besides gram positive, rod shaped and anaerobiotic, the absence of it on the TSC agar could be explained by drawn-out exposure to air during vaccination in the thioglycollate stock, thereby increasing the O degrees and making an unfavourable, aerophilic environment.

Both MSC and RV are selective enrichment stock for the isolation of Salmonella, therefore the turbidness observed in Table 2 due to the bacterial growing. Further vaccination into BSA yielded positive consequences of black mettalic settlements surrounded by black percipitate but negative for E.coli. Due to bismuth sulfite and superb green that permit the selective growing of Salmonella but inhibits Enterobacteiace ( E.coli ) . Furthermore, XLD indicated the presence of Salmonella as it was able to decarboxylate lysine, cause an alkalic reaction and bring forth sulfide from the thiosuphate of the medium, the black centered ruddy settlements are a consequence of sulfide responding with an Fe salt. The presence of level bluish settlements on the CLED medium was characteristic of Salmonella, the medium clearly differentiates the micro-organism found in piss, the presence of xanthous settlements in the meat sample indicated taint from either E.coli or S. aureus. Meanwhile, SIM medium was used to distinguish enteral Bs based on its sulphide production which turned the agar black, indole negativeness and motility. TSI was the index medium that contained glucose, lactose, sucrose and ferric sulphate to categorise Salmonella spp. and other enteral bacteriums. H2S production caused the darkening of the medium.

The presence of Escherichia coli was determined through trials such as the presence of gas bubbles in the LT and EC stock, the former had Na lauryl sulfate as the selective agent for coliform bacteriums and Durham tubes detected gas production ; while the lactose content of the latter favoured the growing of coliform bacteriums that metabolize lactose with gas formation and the gall salts inhibited the growing of gm positive bacteriums. Furthermore, SIM medium was used to corroborate the enteral being based on its sulphide production which turned the agar black, indole positiveness and motility.

Staphylococcus aureus was detected from the meat sample, as the BP agar exhibited balck settlements with a clear zone of proteolysis, drawn-out incubation yielded a more opaque zone. The presence of glycine and pyruvate favored S. aureus growing while the Li and tellurite inhibited viing bacteriums. The decrease of tellurite resulted in balck settlements and the proteolysis of egg protein made a clear zone that surrounded the settlements, farther incubation caused an opaque zone due to lipases. Catalase trial was positive but the coagualse trial was inconclusive, likely due to other bacterial growing on the medium.

The presence of Pseudomonas spp. was confirmed with the presence of growing on the Pseudomonas Agar, as any growing on the media would bespeak the species, and the green settlements further indicated presumptive Pseudomonas aeruginose growing. Plate count agar ( PCA ) nevertheless was used to recite the entire feasible cell count, the settlements were over 300 and excessively legion to number.

For the dairy merchandise, Listeria monocytogenes was detected in the cheese sample. In LSA, aesculin was added as a differential index, hence it gets hydrolysed and reacts with the Fe salt to give a black precipitate around the settlements. Both TYSEA and TYSEB contributed to the verification of L. monocytogenes. Positive catalase trial and the characteristic toppling motility of the bacteria were observed. In add-on to its gm positive, facultative anaerobic and non-sporing atributes. Listeriosis is one of the prima causes of decease in foodborne illnesess, therefore accurate testing and bar is needed as it remains one of the most unsafe jeopardies in the doof industry.

Bacillus Cereus was present in the flour sample, a finished cereal merchandise. Gram positive, facultative B. Cereus is harmful as it causes minor foodborne unwellnesss that have diarrheal and emetic symptoms. Mold growing was obserbed as barm infusion stimulated the growing of casts in OGYE Agar Base, with dextrose as the C energy beginning while Terramycin inhibited bacterial growing. The PEMBA medium turned from green to blue due to bromothymol blue that acted as a pH index, B. Cereus can be determined in the medium due to precipitation from lecithin hydrolysis. The vegetive cells in B. Cereus imparted heat opposition to the miroorganism, hence when cooked nutrient is refrigerated improperly, the endospores would shoot and ensue in the production of enterotoxins, extremely heat immune and pH resistant.

In the domestic fowl sample Campylobacter jejuni was absent from both the mention and sample. The consequence was unexpected as Campylobacter is rather prevailing in domestic fowl, due to traverse taint from extremely colonized countries in the animate being ‘s GI piece of land. This was likely due to incorrect vaccination techniques such as drawn-out exposure of civilization to the air, which increased O degrees. As the rod shaped, non sporing bacteria is microaerophilic in nature, in demands an environment which containins lower concentration of O than that of the ambiance. This bacteria represents one of the most common causes of human stomach flu.

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