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Aerobic And Anaerobic Examination Of Samonella Biology Essay

The genus Salmonella belongs to the family Enterobacteriaceae. As the name implies, Enterobacteriaceae is comprised of bacteria that proliferate in the intestine. Salmonella is a gram- negative flagellated rod- shaped bacterium. Salmonellae are facultative anaerobes with both respiratory and fermentative metabolic pathways. They are oxidase negative, ferment glucose and produce acid and gas, grow on citrates as a sole source of energy, decarboxylate lysine and ornithine, generally produce hydrogen sulfide, and do not hydrolyze urea. One of the characteristic of this genus is that most members do not ferment lactose or sucrose.

10The statistically based sampling scheme required to ensure ‘absence’ of Salmonella is extensive and requires a very significant laboratory input. This method would be used in ensuring adequate processing in combination with simple and reliable methods of verification. A three step methods is required involving pre- enrichment (resuscitation) and selective enrichment, isolation steps and identification tests.

1In pre- enrichment (resuscitation) and selective enrichment step, while isolation of Salmonella from clinical samples often requires only direct plating on selective agar media, detection of the pathogen in food necessitates an enrichment process to increase the numbers before isolation and identification can be accomplished. Salmonella in food is often present in an injured state. Enrichment steps are normally designed to resuscitate these injured Salmonellae and increase their number to detectable levels. In the pre- enrichment step, the food sample is mixed with a suitable nonselective medium and the mixture is incubated. Lactose broth is the most commonly used pre- enrichment medium, in spite of the fact that most Salmonella isolates do not use lactose as a carbon source. Selective enrichment includes sub culturing the pre- enrichment into tubes containing selective broth. Selenite cystine broth and tetrathionate broth are widely used for selective enrichment.

Isolation includes streaking enrichments onto selective- differential agar media and recognizing presumptive Salmonella colonies on the incubated plates. Xylose lysine deoxycholate(XLD) agar and Hektoen enteric (HE) agar are commonly used for isolation of Salmonella from enrichments. These agents inhibit gram- positive and nonenteric bacteria. Additionally, bismuth sulfide (BS) agar is an ideal medium for isolating Salmonellae. It has high selectivity for the bacterium and allows detection of small levels of H2S generated by isolated colonies.

Lastly, identification tests rely on biochemical and serological identification. Biochemical identification is a conventional detection method. Decarboxylation of lysine in LIA, fermentation of glucose anaerobically in TSI agar, production of H2S in TSI and LIA media, and utilization of citrates are important biochemical properties for identification of Salmonella isolates. While isolates that produce typical reactions by biochemical testing should be confirmed as Salmonella by serological analysis.

Material:

Fresh chicken sample, buffered peptone water, mannitol selenite cystine broth, RV medium, dried XLD and bismuth sulfite agarLysine decarboxylase broth, decarboxylase broth base, ONPG broth, CLED medium, two nutrient agar slopes, inoculating loop, wax pensil, Stomacher, autoclave

Methodology :

As stated in the lab manual.

Results :

Selective enrichment)

Culture Medium

Observations

Interpretation/ Conclusion

Rappaport-Vassiliadis (RV) Broth

The initial blue-green colour became lighter and turbidity occurred

Salmonella or other organisms may present in this medium

Mannitol Selenite Cystine (MSC) Broth

The color of medium changed to deeper amber (orange)

Salmonella or other organisms may present in this medium

Plating the Isolated Salmonella on Selective Agar Plate

Culture medium

Culture sources

Observations

Interpretation/ Conclusion

Xylose-Lysine-Desoxycholate (XLD) Agar

RV Broth

Colonies have the same color as the culture medium (red), translucent, with a black centre

Salmonella may be present

MSC Broth

Colonies have the same color as the culture medium (red), translucent, with a black centre

Salmonella may be present

Bismuth Sulphite Agar (BSA)

RV Broth

Metallic sheen black and green colonies in heavy growth

Salmonella or H2S negative Salmonella may be present

MSC Broth

Metallic sheen black and green colonies in growth

Salmonella or H2S negative Salmonella may be present

Biochemical Confirmation

Culture sources

Purple Lysine Decarboxylase Broth

CLED Agar(Blue)

Triple Sugar Iron (TSI )Agar Slant (Red )

RV Broth-XLD Agar

Purple(+)

Yellow colonies, Agar remains blue

Yellow slant (acid), cracked and yellow butt (acid and gas), No black agar (No H2S)

RV Broth- BSA

Purple(+)

Yellow colonies, yellow agar

Yellow slant (acid), cracked and black butt (gas), black agar(H2S)

MSC Broth-XLD Agar

Yellow(-)

Deep blue flat, Agar remains blue

Red slant (alkaline), cracked and black butt (gas), Black agar(H2S)

MSC Broth-BSA

Purple(+)

yellow colony, yellow agar

Yellow slant (acid), cracked and black butt (gas), Black agar(H2S)

Discussions :

In this experiment, we use chicken meat as the sample to do the examination of present for Salmonella. In the beginning, buffered peptone water was used for resuscitation which was pre-enrichment the Salmonella from the food sample. It will help in recovery of Salmonella. The purpose of use buffered peptone water is providing conditions for resuscitation of cells that have been injured by process of preservation. So, small number of Salmonella may have their generation time increased because of competitive growth and may not reach the minimum number for successful isolation.

After 20hours of incubation under 37ºC, the resuscitation fluid was transfer into selective enrichment media which were mannitol selenite cystine broth and Rappaport-Vassiliadis, RV medium. 7RV medium was used for the selective enriching of Salmonella which contained tryptone as the sources of growth. On the other hand, mannitol selenite crystine broth allowed the fermentation of Salmonella. Therefore both medium acted as enrichment agent. From the result showed above, in these two medium, both showed colour changes and development of turbidity occurred. This indicated the Salmonella might present in the broths.

For next steps, xylose lysine desoxycholate (XLD) and bismuth sulfite agar (BSA) were used for selective agar media. 6XLD medium was selective, differential medium which inhibitory to gram-positive organism. selective agar media. XLD medium was selective, differential medium which inhibitory to gram-positive organism. It was formulated for the isolation and presumptive identification of both Salmonella and Shigellae. In primary differentiation of Salmonella and Shigellae for non-pathogenic bacteria, it will rely on the xylose fermentation. 8Most bacteria, including Salmonella, can ferment the sugar xylose to produce acid whereas for Shigella colonies, it cannot ferment it and therefore remain red. After exhausting the xylose supply Salmonella colonies will decarboxylate lysine, increasing the pH once again to alkaline and mimicking the red Shigella colonies. Salmonellae metabolise thiosulfate to produce hydrogen sulfide, which leads to the formation of colonies with black centers and allows them to be differentiated from the similarly coloured Shigella colonies. From the result, it showed XLD medium had indicate the growth of Salmonella from both enrichment media. Colonies had the same color as the culture medium (red), translucent, with a black centre.

9Bismuth Sulphite Agar is a selective medium for the isolation and preliminary identification of Salmonella typhi and other salmonellae from pathological material, sewage, water supplies, food and other products suspected of containing these pathogens. It is particularly useful for the isolation of lactose-fermenting salmonella. 5The growth of Salmonella indicated by the production of black colonies and metallic sheen surrounding the colonies. Sulphur compounds provide a substrate for hydrogen sulphide production, whilst the metallic salts in the medium stain the colony and surrounding medium black or brown in the presence of hydrogen sulphide. Bismuth sulfite agar should not be used after storage for more than 24 – 36 hours. The reason is when this media becomes green, it is too inhibitory, and one cannot expect good result.

Next, three confirmation tests were undergo in order to determine the Salmonellae which were Purple Lysine Decarboxylase Broth, CLED Agar (Blue), and Triple Sugar Iron (TSI )Agar Slant (Red). 4The lysine decarboxylase broth contains glucose, L-lysine and bromocresol purple. During the initial state of incubations, fermentation of glucose will produce acid result in change colour in bromocresol to yellow. The ability of Salmonella to decarboxylase lysine to cadaverine cause the color changed back to purple. From above result, except for MSC broth- XLD agar, other three showed positive test to lysine decarboxylase broth. This showed the reaction of typical Salmonella.

Besides, CLED Agar was used to identify Salmonella. 3CLED medium was used in urinary bacteriology, which supporting the growth of all urinary pathogens and giving good colonial differentiation and clear diagnostic characteristics.The Salmonella produced flat blue colonies in the MSC broth-XLD agar. Another confirmation test was TSI agar test. This medium differentiates gram negative bacteria based on their fermentation of lactose, dextrose and sucrose as well as production of hydrogen sulfide. When these carbohydrates were fermented, the medium will change from red to yellow indicated the production of acid. Sodium thiosulfate was reduced by hydrogen sulfide. The hydrogen sulfide will react with iron salt to yield black iron sulfide. Except for RV broth-XLD agar, all other three showed positive result. This showed the present of Salmonella as gram negative organism.

Conclusion :

After various procedures and tests, Salmonella sp was identified present in the chicken sample. This showed the raw chicken contains microorganism which was harmful for human health. Proper handling should be applied to produce safety food.


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