Laboratory Activity 2: Media Preparation and Aseptic Technique PDF

Polytechnic University of the Philippines College of Science Department of Biology MICROBIOLOGY AND PARASITOLOGY (LABORATORY) BIOL 014 Laboratory Discussion 3: Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology Trisha Mae M. Cantillano ...

Polytechnic University of the Philippines College of Science Department of Biology MICROBIOLOGY AND PARASITOLOGY (LABORATORY) BIOL 014 Laboratory Discussion 3: Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology Trisha Mae M. Cantillano  What is a Culture Media? T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Culture Media and Its Classifications Consistency Nutritional Functional Use Component ✓Liquid media ✓Simple media ✓Enriched media ✓Solid Media ✓Complex media ✓Selective media ✓Semi Solid Media ✓Synthetic or ✓Differential media chemically ✓Transport media defined media T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Culture Media and Its Classifications Classification Based on Consistency Liquid Solid Semi-Solid T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Culture Media and Its Classifications Classification based on Nutritional Component Simple media such as peptone water, nutrient agar can support most non-fastidious bacteria. It is also called as basal media. Complex media contains special ingredients in them for the growth of microorganism. Synthetic media/ Chemically defined media are specially prepared media for research purposes where the composition of every component is well known. It is prepared from pure chemical substance. T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Culture Media and Its Classifications Classification based on Functional Use T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Culture Media and Its Classifications Classification based on Functional Use T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation To prepare media, first, make all necessary computations! ❑Calculate the final volume of the medium that you will use. ❑Take note that for each plate, it must contain about 15 ml of the media, and for each tube, it must contain at least 5 ml of the media ❑Thus, if you want to make 18 plates and 18 tubes…. (18 plates)(15 ml) = 270 𝐦l (18 tubes)(5 ml) = 90 𝐦l ❑= 360 ml T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation Check the label of the media to be used It is important to check the label to (1) know if we can still use the media; and to (2) know its composition T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation Calculate the needed grams For Water Agar, we usually dissolve 20g/L. 20 𝑔 𝑥 = 1000 𝑚𝑙 360 𝑚𝑙 (20𝑔)( 360 𝑚𝑙) 𝑥= 1000 𝑚𝑙 𝒙 = 𝟕. 𝟐 𝒈 T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation Prepare the rest… Weigh the agar powder based on your specific needs (7.2 g of water agar for 360 ml solution). Measure the volume of distilled water that you need (360 ml) Add the measured agar powder into the Erlenmeyer flask with water. Stir the mixture Place the container on a hot plate while stirring continuously until the solution is clear and the agar is dissolved. T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation Prepare the rest… Autoclave the mixture under 121 °C, 15 psi for 15-20 minutes Pour the sterile solution into sterile petri dishes. Allow the agar solution to cool and solidify in the container at room temperature. T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation Nutrient Agar and Potato Dextrose Agar For Nutrient Agar, we usually dissolve 28g/L For Potato Dextrose Agar, we usually dissolve 39g/L 28 𝑔 𝑥 39 𝑔 𝑥 = = 1000 𝑚𝑙 360 𝑚𝑙 1000 𝑚𝑙 360 𝑚𝑙 (28𝑔)( 360 𝑚𝑙) (39𝑔)( 360 𝑚𝑙) 𝑥= 𝑥= 1000 𝑚𝑙 1000 𝑚𝑙 𝒙 = 𝟏𝟎. 𝟎𝟖𝒈 𝒙 = 𝟏𝟒. 𝟎𝟒𝒈 Just repeat the steps… T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation MacConkey Agar Preparation Dissolve 52g of MacConkey agar powder in 1000ml of distilled water 52 𝑔 𝑥 = 1000 𝑚𝑙 360 𝑚𝑙 (52𝑔)( 360 𝑚𝑙) 𝑥= 1000 𝑚𝑙 𝒙 = 𝟏𝟖. 𝟕𝟐𝒈 Just repeat the steps… T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation What if you have to make the media from scratch? Calculate the needed grams for each component: Casein digest peptone 10 𝑔 𝑥 = 1000 𝑚𝑙 360 𝑚𝑙 𝑥 =? 𝑔 𝑜𝑓 𝑝𝑒𝑝𝑡𝑜𝑛𝑒 Yeast extract =?g NaCl =?g Agar =?g T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation For synthetically made media Some media requires addition of antibiotics. For example, rifampicin can be added. Using the stock concentration indicated in the container of the antibiotic and your desired concentration of that antibiotic, compute for the needed volume of the stock concentration of rifampicin that you will add to the culture media T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation For synthetically made media If the stock concentration of your antibiotic in the laboratory is 50 mg/ml, and your desired final concentration of antibiotic is 100µg/ml, you should: 1. Convert the stock concentration into µg/ml 50 𝑚𝑔 1000µg = 50,000 µg/ml 𝑚𝑙 1 𝑚𝑔 2. Compute for the needed volume of stock conc. Of the antibiotic that should be added to the culture media. 𝑪𝟏 𝑽𝟏 = 𝑪𝟐 𝑽𝟐 Where: 𝑪𝟏 = 𝒔𝒕𝒐𝒄𝒌 𝒄𝒐𝒏𝒄. 𝑪𝟐 = 𝒅𝒆𝒔𝒊𝒓𝒆𝒅 𝒇𝒊𝒏𝒂𝒍 𝒄𝒐𝒏𝒄. 𝑽𝟐 = 𝒇𝒊𝒏𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒕𝒉𝒆 𝒄𝒖𝒍𝒕𝒖𝒓𝒆 𝒎𝒆𝒅𝒊𝒂 𝑽𝟏 = 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒔. 𝒄. 𝒕𝒉𝒂𝒕 𝒔𝒉𝒐𝒖𝒍𝒅 𝒃𝒆 𝒂𝒅𝒅𝒆𝒅 𝒕𝒐 𝒕𝒉𝒆 𝒄𝒖𝒍𝒕𝒖𝒓𝒆 𝒎𝒆𝒅𝒊𝒂 T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Media Preparation For synthetically made media If, 𝐶1 𝑉1 = 𝐶2 𝑉2 50,000 µg/ml 𝑉1 = 100µg/ml 360 𝑚𝑙 𝑉1 = ? 3. Once done, just add the computed V1 to the 360 ml of LBA Note that: Antibiotics are filter-sterilized before adding to the culture media T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Mixed and Pure Cultures Mixed Cultures are cultures that has more than one type of organism in a culture media while Pure Cultures contain only a single kind of organism in a media. T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Isolation of Microorganisms Isolation is the process of transferring a single colony from a mixed culture in order to obtain a pure culture. T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Aseptic Technique Aseptic techniques are important microbial techniques being carried to avoid contamination of the workplace, samples and even the person. T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Aseptic Technique T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Aseptic Technique T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Aseptic Technique T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Different Ways to Inoculate and Isolate Microorganisms Serial Dilution Streak Method Stab Culture Subculturing Pour and Spread Plate T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology https://www.youtube.co m/watch?app=desktop& v=MDowwItwcRg https://www.youtube.co m/watch?v=0heifCiMbfY  Factors that affect microbial growth Food Nutrients needed to survive nutrients for microbial growth Moisture Acidity Know the alkalinity and acidity must be in a soluble form tolerance of the organism Microorganisms have different Some organism can’t grow Oxygen Temperature optimum, maximum, minimum with the presence of oxygen temperature Time How long are we going to incubate? T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Cultural Charatceristics We can distinguish items, things, people, and even microorganisms because they have certain characteristics. T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  What is cultural characteristics? A cultural characteristics, also called colonial morphology, is the macroscopic appearances or growth characteristic of a particular culture on a medium. Colony Characteristics on an Colony Characteristics on an Colony Characteristics on Broth Agar Plate Agar Slant T.M.M. Cantillano Culture Media Preparation, Isolation of Microorganisms, and Colony Morphology  Cultural Characteristics Agar Plate Size is the diameter of the colony; can also use large (>1 mm), medium (=1 mm), small (