Food Microbiology Laboratory question, **********Question********** (PLease help
ID: 208475 • 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 what is going on in this lab, 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
Buffered Peptone Water is used for the non-selective pre-enrichment of Salmonella spp. from food. Buffered Peptone Water maintains a high pH over the pre-enrichment period and allows in repair of injured cells that may be sensitive to low pH.
Ingredients: Peptone, NaCl, phosphate
2. Tetrationate (TT) Broth Base
Tetrathionate Broth Base is used as a selective enrichment for the cultivation of Salmonella spp. that may be present in small numbers and compete with intestinal flora. Salmonella organisms may also be injured in food-processing procedures, which include exposure to low temperatures, sub-marginal heat, drying, radiation, preservative, and sanitizers. Salmonella spp. cause many types of infections, from mild self-limiting gastroenteritis to life-threatening typhoid fever.
Mueller demonstrated the effectiveness of Tetrathionate Broth for enriching typhoid and paratyphoid bacilli while inhibiting coliform organisms. Using modified Mueller’s broth, Kauffmann increased the number of positive isolates. Tetrathionate Broth was used in studies for the poultry industry and in a collaborative study for rapid screening of Salmonella in food. Tetrathionate Broth Base, abbreviated as TT Broth Base, is specified in standard methods for Salmonella testing. The FDA Bacteriological Analytical Manual incorporate Tetrathionate Broth Base as a pre-enrichment medium for detecting Salmonella in food materials.
The formula of TT broth can be seen from the attached table. Function of each ingredient is as follows: Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue provides nitrogen, carbon, vitamins, and amino acids in Tetrathionate Broth Base. Selectivity is accomplished by the combination of Sodium Thiosulfate and tetrathionate, which suppresses commensal intestinal organisms. Tetrathionate is formed in the medium upon addition of the iodine and potassium iodide solution. Organisms containing the enzyme tetrathionate reductase will proliferate in the medium. Bile Salt, a selective agent, suppresses coliform bacteria and inhibits Gram-positive organisms. Calcium Carbonate neutralizes and absorbs toxic metabolites
3. XLT4 Agar Base with XLT4 Agar Supplement
Overgrowth of nuisance or contaminating organisms can be a major problem when recovery of a specific organism or species is desired. This is particularly true of Salmonella isolation media where overgrowth of Proteus, Providencia and Pseudomonas can dramatically interfere with the detection and isolation of Salmonella. In 1990, Miller and Tate described a new medium, XLT4 Agar, for isolating Salmonella. Miller and Tate evaluated the sensitivity of the medium by detecting and isolating Salmonella using fecal-contaminated farm samples containing high numbers of competing bacteria. In proceeding studies, Miller and Tate reported that XLT4 Agar significantly improved the recovery of non-typhi Salmonella from chicken and farm environmental drag-swab samples. XLT4 Agar can be used clinically to screen stool samples for non-typhoid Salmonella.
The formula of XLT4 can be seen from the attached table. Function of each ingredient is as follows: Enzymatic Digest of Animal Tissue is the source of complex nitrogen compounds in XLT4 Agar. Yeast Extract is added as a source of vitamins and cofactors. Differentiation of Salmonella from other organisms that also grow on this medium is based on fermentation of Xylose, Lactose and Sucrose, decarboxylase of Lysine, and the production of hydrogen sulfide. Hydrogen Sulfide production is detected by the addition of ferric ions. Sodium Thiosulfate is added as a source of inorganic sulfur. Sodium Chloride maintains the osmotic balance of the medium. Phenol Red is added as an indicator of pH changes, resulting from fermentation and decarboxylation reactions. Agar is the solidifying agent. XLT4 Supplement is added to inhibit growth of non-Salmonella organisms. Typical Salmonella colonies (H2S-positive) appear black or black-centered with a yellow periphery after 18 -24 hours of incubation. Upon continued incubation, the colonies become entirely black or pink to red with black centers. Colonies of H2S-negative Salmonella strains appear pink-yellow.
4. Brilliant green agar
Brilliant Green Agar is a useful medium for Salmonella isolation. Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue provide sources of nitrogen, amino acids, and carbon. Yeast Extract supplies vitamins required for organism growth. Sodium Chloride maintains the osmotic balance of the medium. Lactose and Sucrose are the carbohydrates in the medium. Brilliant Green inhibits grampositive bacteria and most gram-negative bacilli other than Salmonella spp. Phenol Red is the pH indicator and turns the medium yellow with the formation of acid when lactose and/or sucrose is fermented. Agar is the solidifying agent. Typical Salmonella spp. colonies are opaque and pink. The few lactose and/or sucrose fermenting organisms that grow are readily differentiated due to formation of green colonies.
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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.
9. PCR Confirmation Polymerase Chain Reaction (PCR) is a powerful method for amplifying particular segments of DNA, distinct from cloning and propagation within the host cell. This procedure is carried out entirely biochemically, that is, in vitro. PCR was invented by Kary Mullis in 1983. He shared the Nobel Prize in chemistry with Michael Smith in 1993. PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase adds nucleotides to the 3' end of a custom-designed oligonucleotide when it is annealed to a longer template DNA. Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that contains a region complementary to the oligonucleotide, DNA polymerase can use the oligonucleotide as a primer and elongate its 3’ end to generate an extended region of double stranded DNA. 1. Denaturation The DNA template is heated to 94° C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA. 2. Annealing The mixture is cooled to anywhere from 50-70° C. This allows the primers to bind (anneal) to their complementary sequence in the template DNA. 3. Extension The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template. With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.
9.1. InvA gene is Salmonella specific. Confirmation of isolates by PCR amplification of invA gene is a rapid and most accurate way for Salmonella confirmation. In this lab, we will use PCR method to confirm both Salmonella isolates and Vibrio isolates from next lab. The specific gene for Vibrio parahaemolyticus is tlh gene.
10. Agarose Gel Electrophoresis
Principle:
Gel electrophoresis is used to separate DNA fragments including PCR products according to their size and charge.
The product is loaded on the gel that helps DNA filter through itself.
DNA is negatively charged because of the phosphate back bone.
Different sizes of these fragments will cause variation in migration through the gel, allowing smaller fragments to move faster than the larger ones.
Two types of gels can be used for gel electrophoresis; Agarose gel and acrylamide. To visualize the PCR product, DNA gel electrophoresis will be used.
Application:
Estimate DNA molecule size after restriction enzyme digestion (RFLP).
Check PCR amplified product and DNA purification.
Preparation of DNA to be used in other techniques such as Southern blotting.
Agarose:
It's extracted from seaweed and consists of tiny pores allowing the smaller fragments of DNA to migrate faster and farther to anode (+ve). Larger fragments face more friction while migrating through the gel, therefore remaining closer to the cathode (-ve).
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Results:
Formula /l Liter Enzymatic Digest of Casein Enzymatic Digest of Animal Tissue. Bile Salts Calcium Carbonat. Sodium Thiosufate. Final pH: 8.4 ± 0.2 at 25°C Supplement (95221 odine-Potassium lodide Solution Composition per 20.0 mL KI 5.0g lodine 6.0 g 2.5g 2.5g 10 30 Com position of TT brotnExplanation / Answer
Ans-1-Product Summary and Explanation:-
Edel and Kamelmacher1
found that food preservation techniques involving heat, desiccation, preservatives,
high osmotic pressure, or pH changes cause sublethal injury to Salmonella spp. Pre-enrichment in a non-
selective medium allows for repair of cell damage and facilitates the recovery of Salmonella. Lactose Broth is
frequently used for this purpose, but it may be detrimental to recovering Salmonellae.
2 Buffered Peptone
Water (ISO), pH 7.0, maintains a high pH over the pre-enrichment period, allowing repair of injured cells that
may be sensitive to a low pH.3 The pH factor is particularly important for vegetable specimens, which have a
low buffering capacity. In addition, this product is used as a diluent for all general microorganisms and for
Listeria monocytogenes and must be able to support the survival of microorganisms without undue
multiplication or reduction during the period of contact before plating onto agar or inoculation into liquid
media. Buffered Peptone Water conforms with the formula and performance specified in International
Organization for Standardization (ISO), ISO/TS 11133-2014(E).
4,5
Principles of the Procedure
Peptone is the nitrogen, carbon, vitamin, and mineral sources in Buffered Peptone Water (ISO), pH 7.0.
Sodium Chloride maintains the osmotic balance. Disodium Phosphate and Monopotassium Phosphate are
the buffering agents in this medium.
Formula / Liter
Peptone.....10.0 g
Sodium Chloride .. 5.0 g
Disodium Phosphate 3.5 g
Monopotassium Phosphate .... 1.5 g
Equivalent to 9.0 g of disodium hydrogen phosphate dodecahydrate
Final pH: 7.0 ± 0.2 at 25°C
Formula may be adjusted and/or supplemented as required to meet performance specifications.
Precautions:-
1. For Laboratory Use Only.
2. IRRITANT. Irritating to eyes, respiratory system, and skin.
Directions:-
1. Dissolve 20 g of the medium in one liter of purified water.
2. Heat with frequent agitation to completely dissolve the medium, if necessary.
3. Autoclave at 121°C for 15 minutes.
Quality Control Specifications
Dehydrated Appearance: Powder is homogeneous, free flowing, and off white to light beige.
Prepared Appearance: Prepared medium is clear with no to light precipitate, pale to light yellow.
Expected Cultural Response: Cultural response in Buffered Peptone Water (ISO), pH 7.0 incubated
aerobically at 35 ± 2°C and examined for growth after 18 - 24 hours incubation when used as a preenrichment for Salmonellae and Enterobacteriaceae. When used as a diluent; the inoculum must remain
within plus or minus 30% of the starting inoculum after standing for a specified time and temperature..
MICROORGANISM ATCC
APPROX.
INOCULUM
(CFU) EXPECTED RESULTS ACTUAL RESULTS
Pre-enrichment for Salmonella and Enterobacteriaceae detection
Escherichia coli 25922 10-300 Growth
Salmonella choleraesuis 13076 10-300 Growth
Salmonella typhi 19430 10-300 Growth
Salmonella typhimurium 14028 10-300 Growth
Diluent for enumeration of microorganisms and L. monocytogenes
Escherichia coli 8739 10^4 T1 plate counts w/in ± 30%
of counts for T0
Staphylococcus aureus 25923 10^4 T1 plate counts w/in ± 30%
of counts for T0
Listeria monocytogenes 13932 10^4 T1 plate counts w/in ± 30%
of counts for T0
The organisms listed are the minimum that should be used for quality control testing.
Test Procedure
Refer to appropriate references for specific procedures using Buffered Peptone Water (ISO), pH 7.0.
4,5
Results
Growth is indicated by turbidity in the pre-enrichment test.
For the diluent test, plated media is counted and meets the specification stated in the table above.
Storage
Store sealed bottle containing the dehydrated medium at 2 - 30°C. Once opened and recapped, place
container in a low humidity environment at the same storage temperature. Protect from moisture and light by
keeping container tightly closed.
Expiration
Refer to expiration date stamped on the container. The dehydrated medium should be discarded if not free
flowing, or if the appearance has changed from the original color. Expiry applies to medium in its intact
container when stored as directed.
Limitation of the Procedure
Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this medium.
Packaging
Buffered Peptone Water (ISO), pH 7.0 Code No. 9262A 500 g
9262B 2 kg
9262C 10 kg
9262D 50 kg