Lab #9. Introduction. Class samples:

Transcription

1 Lab #9 Introduction Food-borne illness is largely caused by the presence of bacteria in red meat. However, much of these harmful bacteria can be destroyed and prevented by sanitation and safe cooking practices. This lab will establish the presence of microbes in variations of cooking practices and evaluate the effective sanitation of the cutting board utilized. Materials & Methods Experiment: Microbial Analysis of Meat Class samples: 1. Fresh medium rare 2. 2 day old medium rare 3. Expired - well 4. Expired medium rare Weigh 1 gram of beef meat from samples provided, place raw meat on specified and labeled side of cutting board, cook 2 day old meat to medium rare, place cooked meat on opposing labeled side of cutting board. Part 1: Cooked Meat Analysis Place 1 gram cooked meat, broken into pieces, into test tube of the original dilution (10⁰) with 10 ml of MRD solution and shake to mix. Place 9.9 ml MRD in following 5 test tubes to produce serial dilutions of 10 ², 10 ⁴, 10 ⁶, 10 ⁸, and 10 ¹⁰ by transferring 0.1 ml from original (or previous dilution tube to inoculated) via a µl pipette. Using the spread plate method, 1 ml is transferred from correlating dilution to labeled plates as follows: Spread Plates of 2 Day Old Meat Cooked to Medium-Rare Medium Dilutions NA 10 ² 10 ⁴ 10 ⁶ 10 ⁸ 10 ¹⁰ EMB 10 ² 10 ⁴ 10 ⁶ 10 ⁸ BP 10 ² 10 ⁴ 10 ⁶ MYP 10 ² 10 ⁴ 10 ⁶ MRS 10 ² 10 ⁴ 10 ⁶ 10 ⁸ Part 2: Cutting Board Analysis Wash cutting board, take a sterile swab moistened with MRD of the cutting board from labeled raw to cooked side. Place swab of potential cutting board microbes into a test tube containing 1 ml of MRD as

2 the original (10⁰) test tube in the series of serial dilutions 10 ¹, 10 ², and 10 ³. The following 3 test tubes of the serial dilution contain 9 ml MRD, and 1 ml transferred from previous dilution test tube. Using the spread plate inoculation method, the following dilution plates were prepared with 1 ml from corresponding dilution test tubes: Part 3: API Test Spread Plates of Cutting Board Swab from Raw to Cooked Meat Medium Dilutions NA 10 ¹ 10 ² 10 ³ VRBG 10 ¹ 10 ² An API test was performed on a light pink microbial colony retrieved from the VRBG spread plate cutting board swab with a dilution of 10 ¹. The procedure followed to perform an API test is described previously in lab 7 under exercise Results API Reagent synonyms & description: Ferric chloride Dropper bottles (unlabeled) Sulfonic acids Nitrate A N,N dimethyl napthylamine Nitrate B Part 1: Cooked Meat Analysis Spread Plates of 2 Day Old Meat Cooked to Medium-Rare EMB Spread Plates: Dilution 10 ² Dilution 10 ⁴

3 Dilution 10 ⁶ Dilution 10 ⁸ BP Spread Plates: Dilution 10 ² Dilution 10 ⁴ Dilution 10 ⁶ MYP Spread Plates: Dilution 10 ² Dilution 10 ⁴ Dilution 10 ⁶

4 MRS Spread Plates: Dilution 10 ² Dilution 10 ⁴ Dilution 10 ⁶ Dilution 10 ⁸ NA Spread Plates: Dilution 10 ² Dilution 10 ⁴ Dilution 10 ⁶

5 Dilution 10 ⁸ Dilution 10 ¹⁰ Part 2: Cutting Board Analysis Spread Plates of Cutting Board Swab from Raw to Cooked Meat VRBG Spread Plates: Dilution 10 ¹ Dilution 10 ² NA Spread Plates: Dilution 10 ¹ Dilution 10 ² Dilution 10 ³

6 Class Plate Counts: Group 1 Group 2 Group 3 Group 4 Meat/ Cooked Fresh - Medium Rare 2 Day - Well Done 2 Day - Medium Rare Expired- Medium Rare Medium Dilution CFU Dilution CFU Dilution CFU Dilution CFU Plate Plate Plate Plate MYP 10 ² 194 NG NG 10 ⁶ ² 2 BP NG NG NG NG NG NG NG NG EMB 10 ⁸ 134 NG NG 10 ⁴ ² 38 MRS 10 ⁶ 45 NG NG 10 ⁴ ² 36 NA Not Not NG NG 10 ⁶ ² 126 Countable Countable Swab Wood Wood Plastic Plastic NA NG NG 10 ¹ 123 Not Not 10 ¹ 24 Countable Countable VRBG NG NG NG NG Not Countable Not Countable 10 ¹ 11 NG = no growth Part 3: API Test Part 1: Initial Spontaneous Test Results Part 2: Reagent Test Results

7 Discussions The following selective and differentiating mediums are utilized to try to identify which microbes are present and at what quantity. The following table describes which observations may occur within various types of medium and the common micro-organisms expected to grow. This table will be utilized as a reference to attempt to identify growth on cultured spread plates. All information was retrieved from Medium NA EMB BP MYP MRS VRBG Agar Name Nutrient Agar deman, Rogosa, Sharpe Violet Red Bile Glucose Used For cultivation of a wide variety of microorganisms the cultivation of lactobacilli enumeration of Enterobacteriac eae in foods Possible Observations N/A N/A Refer to previous lab (Lab # 8) Dextrose fermenters produce red Expected Micro-organisms Bacillus subtilis Escherichia coli Salmonella typhimurium Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Lactobacillus casei Lactobacillus fermentum Lactobacillus plantarum Enterobacter Pink colonies w/red aerogenes precipitate Escherichia coli Pink colonies w/red

8 colonies with redpurple precipitate halos in the Pseudomonas Colorless to grey presence of Neutral Red, the ph aeruginosa Salmonella typhimurium Pink colonies w/red Precipitate indicator Staphylococcus aureus N/A *Per the lab syllabus, MRS should grow lactobacilli, and VRBG should grow enterobacteria. Part 1: Cooked Meat Analysis (2 Day Old Cooked to Medium Rare) EMB Spread Plates: The EMB spread plates displayed dense growth, appearing colorless (possibly Pseudomonas aeruginosa or Salmonella typhimurium) covering about 90% of the agar surface with little isolation achieved and few colonies. Growth was uniform within the thick, blanketed areas. The micro-organism likely performs aerobic respiration. The density of growth and amount of surface area displaying growth decreased as the dilution factor increased. Due to the affluent growth, a result can be deduced that EMB supplies nutrients necessary for the growth and isolation of a single species of microbe present in the sample of meat cooked to medium rare. BP Spread Plates: The only BP spread plate to successfully produce growth was a dilution of 10 ² in which the total number of colonies was too few to count with very small diameter and large spacing between colonies. Growth appears black, round and raised (possibly Enterococcus faecalis or Staphylococcus epidermidis). Due to the lack of growth produced upon the BP spread plates, an interpretation can be deduced that BP plates do not provide the necessary nutrients nor has an inhibitory effect upon the microbes found within the cooked meat sample. MYP Spread Plates: The MYP spread plates displayed little colony isolation but rather densely swarming growth of a colorless (possibly Bacillus subtilis) microbe upon the surface area of all dilutions prepared. On one occasion (10 ⁴), the color of ¼ of the agar is observed to have undergone a reaction changing from yellow-orange to red-orange. The microbial growth pattern denotes aerobic respiration. Although Bacillus cereus typically produces pink microbial growth, it may also create a white precipitate from the reaction between lecithinase and the egg yolk within the medium; there exists a white precipitate across the surfaces of dilution plates 10 ² and 10 ⁴. MRS Spread Plates: The MRS plates confirm the presence of lactobacilli within the sample of meat cooked to medium rare by producing microbial growth upon all four dilution plates prepared. MRS plates are utilized for the cultivation of lactobacilli specific micro-organisms; however it is possible to demonstrate the presence of differing types of lactobacilli. The growth upon the MRS plates appeared as isolated opaque white rounded and raised small colonies, as well as a more translucent white branching growth. Total cell

9 growth decreased as the dilution factor was increased, but total growth upon the first dilution plate of 10 ², was too great to be counted. NA Spread Plates: Nutrient agar is utilized to promote the growth of many various micro-organisms rather than inhibiting or limiting specific organisms. The five NA plates produced displayed large variations of growth, possibly indicating a mistake in the dilution process due to a lack of growth upon the first dilution plate which should contain the largest quantity of bacteria; however growth of colonies too numerable to count upon the second dilution spread plate. Colonies upon dilution plate 10 ⁴ appeared with differing small diameters of symmetrical off-white demonstrating a preference of spacing rather than dense clusters, as well as a preference for aerobic metabolism demonstrated by the location at the surface of the medium. The growth upon dilution 10 ⁶ and 10 ¹⁰ appear as thin, translucent film densely swarming rather than preferring spaced colonies. Part 2: Cutting Board Analysis VRBG Spread Plates: The microbial growth upon the VRBG spread plates demonstrate definitively that the sanitation methods performed do not prevent or destroy micro-organisms upon a plastic cutting board. The variation of observed physical traits in growth, including color, shape, both absence and presence of halos and changes to color or reactions with the agar medium may indicate differing species. The VRBG exhibits a large shift in color from a deep opaque maroon to a transparent peach following reactions and nutrient depletion by the large white (surrounded in grey halo) and pink isolated colonies. VRBG agar is utilized to present enterobacteria; however growth present may be Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, or Staphylococcus aureus when compared to traits presented in observational growth described in the above table. NA Spread Plates: The NA spread plates displayed little to no growth. The growth which exhibited upon the agar displayed 3 distinct variations in growth patterns. Upon the 10 ¹ dilution plate, growth appears as extremely small densely clustered off-white colonies or a thin film. Upon the 10 ² dilution plate, 2 single moderately sized tan colonies appear at one end of the agar, and at the other a growth appears with the similarity of a dried, pressed off-white flower. The result of little to no growth is perplexing due to the objective of nutrient agar to support a wide variety of growth and nutrient needs. Class Plate Counts: The results of the class plate counts performed upon the variations of fresh to expired meat cooked to either well done or medium rare are difficult to analyze due to inconsistency of growth. It would have been expected that medium-rare meat would display greater presence of microbe than well-done meat because the extended duration and exposure to heat should destroy more bacteria. Also, it would be expected that expired meat would demonstrate a larger presence of bacteria, unless all nutrients were depleted resulting in cell death.

10 The results depicted by the plate counts performed upon wooden and plastic cutting boards can be interpreted as plastic cutting boards enabling greater growth of bacteria, possibly due to cuts made by knives into the plastic which can harbor growth. The type of wood used may have been antimicrobial or a type which will inhibit the growth of bacteria resulting in no growth upon dilution plates. Part 3: API Test The API test performed upon a colony selected from the VRBG swab dilution plate, demonstrated many positive spontaneous reactions, as well as many reactions to reagents. However, identification could not be made. Attempts were made to input variations of the seven digit code where possible false positive results could have been interpreted, still identification was not possible. API Results & Interpretations Spontaneous Reagent Test Observation Result Observation Result Interpretation Symbol ONPG Off-white Positive Produces Betagalactosidase + ADH Pink Positive Produces arginine + dehydrolase LDC Pink Positive Produces lysine + decarboxylase ODC Red Positive Produce ornithine + decarboxylase CIT Blue Positive Utilizes citrate as sole + carbon source H₂S Clear Negative Does not reduce sulfur - URE Yellow Negative Does not produce urease - TDA Clear Negative Light peach Negative Does not produce - tryptophan deaminase IND Clear Negative Yellow Negative Does not produce indole - VP Clear Negative Pink Positive Produces acetoin +/- (possible contaminant) GEL Black Positive Produces gelatinase + dispersed GLU Blue Negative Yellow Positive Ferments glucose + MAN Blue Negative Does not ferment - mannitol INO Yellow Positive Ferments inositol + SOR Blue Negative Does not ferment sorbitol - RHA Blue Negative Does not ferment - rhamnose SAC Blue Negative Does not ferment sucrose - MEL Blue Negative Does not ferment - melibiose AMY Yellow Positive Ferments amygdalin + ARA Blue Negative Does not ferment -

11 Variations of seven digit code: , arabinose Key Spontaneous reactions occurred Reagents Added

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