Microbiological Analysis of Food Products PDF

# Microorganisms in Food ## Microorganisms in Food A diagram displaying five common microorganisms in food: * **Molds** * **Bacteria** * **Parasites** * **Yeast** * **Viruses** ## Microbiological Analysis of Food Products * **Aim** 1. To determine the total number of microorganisms (bacteria,...

# Microorganisms in Food ## Microorganisms in Food A diagram displaying five common microorganisms in food: * **Molds** * **Bacteria** * **Parasites** * **Yeast** * **Viruses** ## Microbiological Analysis of Food Products * **Aim** 1. To determine the total number of microorganisms (bacteria, yeast & molds) present in food products. 2. To determine the pathogenic bacteria in the selected food products * **Principle** The presence of microorganisms in food may be considered harmful in some cases, while in others it is definitely beneficial. Microorganisms exist virtually everywhere. Left undisturbed in their own habitat, most are beneficial in some way. Some microorganisms have been used successfully to produce our favorite foods, such as yogurt, wine, beer, sauerkraut, buttermilk, vinegar, bread, and cheeses. Generally speaking, however, the unintended introduction of microorganisms to our food (including otherwise beneficial microorganisms is a serious health hazard. For example, Escherichia coli, one of the most common enterics in humans and other mammals, can cause mild to severe illness or even death if ingested. **Materials:** * **Cultures:** Samples of different foods such as vegetables, beef, fruit * **Media:** Brain-heart infusion agar, plate count agar, malt extract agar. ### Procedure 1. Using separate sterile pipettes, prepare decimal dilutions of 10-2, 10-3, 10-4, and others as appropriate, of food homogenate. (For food homogenate: Add 450 ml phosphate-buffered dilution water to blenderjar or stomacher sterile bag containing 50 g analytical food sample and blend for 2 min. This results in a dilution of 10-1. 2. Make dilutions of original homogenate promptly, using pipettes that deliver required volume accurately. 3. Prepare all decimal dilutions with 9 ml of sterile diluent plus 1 ml of previous dilution, unless otherwise specified, by transferring 1 ml of previous dilution to 9 ml of diluent. 4. Pipette 1 ml of each dilution into separate, duplicate, appropriately marked petri dishes. 5. Add 12-15 ml Brain-heart infusion agar or plate count agar, malt extract agar (cooled to 45 ± 1°C) to each plate within 15 min of original dilution. Pour agar and dilution water control plates for each series of samples. 6. Immediately mix sample dilutions and agar medium thoroughly and uniformly by alternate rotation and back-and-forth motion of plates on a flat level surface. 7. Let agar solidify. Invert solidified petri dishes, and incubate promptly for 24 ± 2 h at 37°C. 8. After the pour plates have cooled and the agar has hardened, they are inverted and incubated at 370C for 24 hours. 9. At the end of the incubation period, select all of the petri plates containing between 30 and 300 colonies: * Plates with more than 300 colonies cannot be counted and are designated too many to count (TMTC). * Plates with fewer than 30 colonies are designated too few to count (TFTC). * Count the colonies on each plate. A Quebec colony counter should be used. 10. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to liquified agar. ### Serial Dilution Procedure A diagram containing a series of tubes, demonstrating serial dilution. The following information is given below the diagram: * **Calculation:** Number of colonies on plate x reciprocal of dilution of sample = number of bacteria/ml * **For example:** if 32 colonies are on a plate of 1/10,000 dilution, then the count is 32 x 10,000 = 320,000 bacteria/ml in sample. ### Observations 1. Choose a plate that appears to have between 30 and 300 colonies. 2. Count the exact number of colonies on that plate using the colony counter (as demonstrated by your instructor). 3. Calculate the number of CFUs per ml of original sample as follows: The number of CFUs per ml of sample = The number of colonies (30-300 plate) x ml of dilute sample plated * = Number of colonies * = Dilution factor of plate counted * = Number of CFUs per ml ### Record your results and find the average number of cfus/ ml by adding these results from all of your plates and dividing by the number of plates. | Type of Food | Dilution | Number of Colonies per Plate | Number of Organisms per ml | |--------------|----------|---------------------------|---------------------------| | | | | | | | | | | | | | | | | | | | | | | | | | # **Microorganisms involved in fermented foods** A diagram showing a table with four categories and corresponding microorganisms involved in fermented goods: | Category | Microorganisms | |:--------|:----------------| | Lactic Acid Bacteria | *Lactobacillus, Streptococcus* | | Fungi | *Aspergillus, Penicillium* | | Other Bacteria | *Propionibacterium, Brevibacterium* | | Yeast | *Saccharomyces* | # **Hydrogen Sulphide Test** * **Aim** * To demonstrate the formation of hydrogen sulphide gas using SIM agar medium. * To determine the ability of microorganisms to produce hydrogen sulphide from substrates such as the sulfur containing amino acids or inorganic sulfur compounds. * **Principle** Many proteins are rich in sulfur-containing amino acids such as cysteine. When these proteins are hydrolyzed by some bacteria, the amino acids are released and taken up as nutrients. Cysteine, in the presence of the enzyme cysteinedesulfurase, loses its sulfur atom through the addition of hydrogen from water to form hydrogen sulfide gas. Gaseous hydrogen sulfide may also be produced by the reduction of inorganicsulfur-containing compounds such as thiosulfate (S2O3 2-), sulfate (SO42-) or sulfite (SO3 2-). For example, when certain bacteria take up sodium thiosulfate, they canreduce it to sulfite using the enzyme thiosulfate reductase with the release of hydrogen sulfide gas. Such a reduction occurs during anaerobic respiration in which respiring cells use something other than oxygen (such as thiosulfate) as the final electron acceptor in the respiratory electron transport chain. * **Materials:** * Test organisms, SIM agar tubes and inoculating needle. * **Procedure:** 1. Label each of the SIM agar deep tubes with the name of the bacterium or sample to be inoculated, and date. 2. With an inoculating needle, and using aseptic technique, inoculate each tube with the appropriate bacterium by stabbing the medium. of the way to the bottom of the tube. 3. Be careful when inoculating the tubes to withdraw the needle from the agarin a line as close as possible to the line used when entering the agar. Do not allow the inoculating needle to touch the bottom of the tube. 4. Incubate the cultures for 24 to 48 hours at 37oC. 5. Examine the SIM cultures for the presence or absence of a black precipitate along the line of the stab inoculation. A black precipitate of FeS indicates the presence of H2S. Any blackening of the medium is considered a positive test for H2S production. ### Result & Discussion A table showing the results of the hydrogen sulfide test: | Tested Organism | Color of Medium | H2S Production (+ or -) | |:---------------|:---------------|:---------------------| | | | | | | | | | | | | | | | | A diagram showing two test tubes illustrating the results of the hydrogen sulfide test. One test tube shows no black precipitate. The other shows a black precipitate. * **H2S -ve:** No black precipitate * **H2S +ve:** Black precipitate