Food Microbiology Laboratory question, **********Question********** (PLease help
ID: 208954 • Letter: F
Question
Food Microbiology Laboratory question,
**********Question********** (PLease help me to figure out the answers after reading/ using following text, thx a lot)
1. Deeply introduce this experiment, what methods have been used and why.
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Laboratory: Microbiological analysis of food products (Isolation of Salmonella spp.)
Media used in this Lab
1. Buffered Peptone Water
2. Tetrationate (TT) Broth Base
3. XLT4 Agar Base with XLT4 Agar Supplement
4. Brilliant green agar
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Food products used: pork sample
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Procedure:
Each group will be provided with 1 pork sample bought from local market. After pre-enrichment, selective enrichment, and selective plating, Salmonella spp. will be further confirmed by PCR with Salmonellaspecific invasion protein invA gene. This method allows rapid and sensitive and specific detection of Salmonella spp. in food and clinical samples.
1. Non-selective pre-enrichment
1.1. Put your sample in the stomacher plastic bag.
1.2. Add 50 ml of sterile buffered peptone water.
1.3. Place the bag in the stomacher, mix at medium speed for 30 seconds.
1.4. Pour 20 ml of homogenate into a 50 ml Falcon tube.
1.5. Incubate at 35°C for 18-24 hours.
2. Selective enrichment
2.1. Transfer 1ml of the pre-enrichment to 10ml Tetrathionate broth.
2.2. Incubate at 42°C for 18-24 hours.
3. Spread on selective agar plate
3.1. Pick a loopful of selective enrichment and streak on a XLT4 agar plate.
3.2. Incubate at 35°C for 18-24 hours.
4. Single colony inoculation (Follow-up 3)
4.1. Observe if single colonies form on the plate. Typical Salmonella colonies (H2S-positive) appear black or black-centered with a yellow periphery after 18-24 hours of incubation.
4.2. Pick 4 single suspected colonies (with black centre) from the XLT4 plate and streak on a new XLT4 agar plate, label them as X1/X2/X3/X4. (A total of 4 XLT4 plates)
4.3. Pick 4 single suspected colonies (without black centre) from the XLT4 plate and streak on BGA agar plate, label them as B1/B2/B3/B4. (A total of 4 BGA plates).
4.4. Incubate at 37°C for 18-24 hours. Typical Salmonella colonies (H2S-negative) on BGA appear opaque and pink.
5. Purification on LB agar
5.1. Observe the growth of Salmonella streaked on XLT4 and BGA on previous day. Pick 2 single colonies from the XLT4 and BGA plates which are highly suspected to be Salmonella spp., respectively (label them as X1, X2, B1 and B2 clones) and streak on a LB agar for further experiment. Note the colour of the colonies picked. These four colonies (X1, X2, B1 and B2) will be subjected to further analysis include API20E test and PCR confirmation (A total of 4 LB plates).
5.2. Incubate at 37°C for 18-24 hours.
6. API20E test for Enterobacteriaceae identification
6.1. Part 1: Oxidase Test
6.1.1. Oxidase test must be performed before using the API20E system (API20E system is only working for oxidase-negative bacteria)
6.1.2. Four API20E assays will be provided for each group for 4 colonies picked previously.
6.1.3. Use a loop and pick a well-isolated colony and rub onto a small piece of filter paper
6.1.4. Place 1 or 2 drops of 1% Kovács oxidase reagent on the organism smear.
6.1.5. Observe for color changes.
6.1.6. Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds. Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes.
Part 2: API20E test:
Four API20E strips will be provided for each group.
7. Read API20E result
7.1. For some of the compartments, you can just read the change in color straightway after 24 hours but for some you have to put reagents before reading.
7.2. Add following reagents to these specific compartments
TDA: Put one drop of Ferric Chloride
IND: Put one drop of Kovacs reagent (Read within 2 minutes)
VP: Put one drop of VP reagent 1 (40 % KOH ) and one drop of VP Reagent 2 (-Naphthol)
(Wait for 10 minutes before telling it negative).
7.3. Mark each test whether it is positive or negative on the score sheet according to this below color scale.
7.6. Identify your unknown organism using the 7-digital code via apiweb or API catalog.
8. DNA Extraction from Bacteria
8.1. Label eppendorf tubes with the sample name accordingly.
8.2. Scratch a loopful of cultures from LB plates [4 colonies (X1, X2, B1 and B2) from Salmonella Practical and 4 colonies (S1, S2, S3 and S4) from Vibrio Practical] and resuspend with 500 l 1X Phosphate buffered saline (PBS) in the eppendorf tube.
8.3. Vortex to suspend the culture evenly in the PBS solution.
8.4. Boil the tube at 100°C with a heat block for 5 min.
8.5. Cool the tube down and centrifuge at 13,000 rpm for 2 min.
8.6. Save the supernatant as DNA template for PCR using Eppendorf.
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Results:
stri neck The strip is inoculated with a saline suspension of a pure culture (to rehydrate the wells) Most of the wells would be filled up to the neck Some will be filled up to the top with sterile Top mineral oil well or cupule LDC ODC ADH H2S URE Neck |- fill line for most of the wells Some wells will be filled all the way up to the top CIT IVP lGELExplanation / Answer
the experiment is mainly to find out the species of salmonella.
first, you need to pick up the black isolated colony or black with yellow periphery is selected for XLT4. Pink and opaque colony for BGA.
the then selected colony is further incubated and the most isolated and expected colony is selected for further selection.
then oxidase is conducted. if oxidase test is positive the salmonella will not be present in the sample. this is because bacteria from enterobacteriaceae family shows negative result of oxidase test. since salmonella belongs to the enterobacteriaceae oxidase results the same.
once it is clear that oxidase test is negative then, API20E test is coducted to detect the species of bacteria.
so compare the strip with the list and score to detect the answer
X3 -6704552
X1-6704552
b3-1604572
b2-5004552
so the X1 and 2 is salmonella enterica