Food Spoilage Microorganisms

Food Spoilage Microorganisms B. Materials and Tools Ingredients:  Examples of Food/Culture Group:   5 types of processed food 2 types of putrefactive bacterial cultures Medium/Chemicals:  Grouped (4 tubes/cup each per medium) Medium for proteolytic test:      Skim milk agar (SMA), HCl 1% Nutrient Gelatin (NG) Litmus milk (LM) Medium for amylolytic test: Starch Agar (STA) and iodine solution (lugol) Medium for acid formation test:   Dextrose Tryptone Bromcresol Purple Agar (DTBPA) Methyl-red media – Voges proskauer (MR-VP), methyl red indicator, 5% alphanaphthol, 40% KOH Medium for lipolytic test:       Plate count agar (PCA) + 1% oil + neutral red indicator Medium for pectinolytic test: Polypectate Gel Medium (PGM), HCl 6-8 N Medium for H2S formation test : Sulfite Agar (SA) 2 tubes of 45 mL diluent solution Tool :     Scales Pipettes, petri dishes, tubes, test tubes incubator water bath temperature 50o C C. How it works Material Preparation: For the food to be tested, a 1:10 dilution is carried out using this method weigh 5 g or pipette 5 mL of the material and put it in the diluent solution 45 mL, then stir until evenly mixed. Then let it sit for 5-10 minutes while stirring occasionally to even out the suspension Testing Method: -A total of one eye of culture and food extract is taken and scratched on each each on agar cups SMA, STA, DTBPA, PCA 1% oil and PGM. Every will take the culture and streak it on a different medium, a loop needle should be used burned first until red hot. -A total of 0.1 mL of culture and food extract was pipetted and each was put into in a tube containing NG, LM and MR-VP medium and 1 mL of each each was put into a tube containing liquid SA medium taken from 50o C bath Shake well, then let stand until frozen at temperature, like. -All plates and tubes were incubated for 2-3 days at 28-30o C, except NG medium and MRVP medium whose incubation can be extended up to one weeks if it still produces a negative test. -The characteristics of food spoiling microorganisms are observed as follows: SMA medium – proteolytic colonies form a clear color all around colonies, and if dropped with 1% HCl it will remain clear because it does not occur protein deposition. -Medium NG – organisms that can hydrolyze gelatin to produce gelatin becomes liquid, and the medium will remain liquid even if it is immersed in ice water. -Medium LM – several reactions that may occur are: 1. Separation of whey or coagulation of milk due to the activity of proteolytic enzymes without acid formation so the litmus color remains blue 2. Formation of acid from the breakdown of lactose or clumping milk where litmus reduction occurs so that the color becomes pink 3. Gas formation is characterized by the presence of bubbles -Medium STA – growing colonies are dripped with iodine solution, where the colonies starch breaker will be surrounded by a yellow/brown area due to starch hydrolysis, while the area around the colony that does not break down the starch remains blue in the presence of iodine -Medium DTBPA – colonies of acid-forming microorganisms will be surrounded by an area yellow due to the color change of bromcresol purple indicator at low pH -MR-VP medium – 1-2 mL of growth from MR-VP medium is transferred to in a test tube and given 3 drops of 5% alpha naphthol solution and 40% KOH. The formation of a dark red color indicates the formation of acetyl methyl carbinol (acetoin) by microorganisms. The remaining MR-VP culture was incubated again for up to a week, then dripped with methyl red indicator. Color changes to red indicates acid formation. -Medium PGM – colonies that are not pectinolytic (hydrolyze pectin) will forms a clear area around the colony when dripped with a settling solution polysaccharides, for example 6-8 N HCl solution -Medium PCA + 1% oil – colonies of fat-breaking microorganisms will break down triglycerides become glycerol and fatty acids thereby lowering the pH of the medium, resulting in a red color appearing at the bottom of the colony due to changes neutral red indicator at low pH. -Medium SA – H2S formation is characterized by the appearance of colored colonies black in SA medium