Microscopy and Slide Preparations PDF

Polytechnic University of the Philippines College of Science Department of Biology MICROBIOLOGY AND PARASITOLOGY (LABORATORY) BIOL 014 Laboratory Discussion 2: Microscopy and Slide Preparations Trisha Mae M. Cantillano  Microscopy: Principles behind Microscope...

Polytechnic University of the Philippines College of Science Department of Biology MICROBIOLOGY AND PARASITOLOGY (LABORATORY) BIOL 014 Laboratory Discussion 2: Microscopy and Slide Preparations Trisha Mae M. Cantillano  Microscopy: Principles behind Microscope Microscopes allow the study of structure of living organisms and the discovery of numerous species that cannot be seen by our naked eye The first microscope uses single lens (Sir Antonie van Leewenhoek) which can magnify 300x the size of the organism. The first microscope that uses double lenses was not invented until the late 16th century T.M.M. Cantillano Microscopy and Slide Preparations  Microscopy: Principles behind Microscope T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Light Microscope- use sunlight or artificial light Bright field microscope Dark field microscope Phase contrast Microscope Fluorescence Microscope Electron Microscope – use electrons Transmission Electron Microscope Scanning Electron Microscope T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Bright-Field Microscope Produces a dark image against a brighter background Usually have several objective lenses Can either be Simple or Compound T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Dark-Field Microscope Produces a bright image of the object against a dark background Specimen appears bright against dark background Provides contrast to unstained tissue so living cells can be viewed T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Phase Contrast Microscope Produces a brighter image in a dark background Contrast is due to out of phase rays Allow to see internal cellular components and examine growth of living cells T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Fluorescence Microscope Exposes specimen to ultraviolet, violet, or blue light Samples are usually stained with fluorochromes Shows bright image of the object from the fluorescent light emitted by the specimen T.M.M. Cantillano Microscopy and Slide Preparations  Different Types of Microscopes and Uses Electron Microscope Co-invented by Max Knoll and Ernst Ruska in 1931 Uses a beam of highly energetic electrons to examine objects on a very fine scale Transmission Electron Microscope and Scanning Electron Microscoe T.M.M. Cantillano Microscopy and Microbial Morphology  Microscopy: Principles behind Microscope T.M.M. Cantillano Microscopy and Slide Preparations  Microscopy: Principles behind Microscope Eyepiece/ Ocular lens: 10x Scanning: 4x Low-power Objective: 10x High-Power Objective: 40x Oil Immersion Objective: 100x Linear Magnification = (eyepiece) x (objective) = 10 x 10 = 100 x T.M.M. Cantillano Microscopy and Slide Preparations  Micrometry Micrometry is the science in which we have some measurement of the dimensions of a specimen observed under microscope. Two types – Stage Micrometer (calibrated) – Ocular Micrometer (non-calibrated) T.M.M. Cantillano Microscopy and Slide Preparations  1 Div = 0.01mm = 10 µm 10 Div = 0.1 mm = 100 µm 100 Div = 1 mm = 1000 µm  Micrometry: Calibrating the Ocular Micrometer c = (20 stage units) x (10 µm/stage unit) Calibration constant (c): 30 of ocular units c = (no. of stage units) x (µm/stage unit) = 200 µm/30 ocular units no. of ocular units = 6.67 µm/ ocular unit T.M.M. Cantillano Microscopy and Slide Preparations  Micrometry: Measuring a Specimen Calibration constant (c):6.67 µm/ ocular unit Specimen size = (no. of ocular units) x (c) = (39 ocular units) x (6.67 µm/ ocular unit) = 260.13 µm T.M.M. Cantillano Microscopy and Slide Preparations  Micrometry: Magnification of Illustration MI = Size of illustration. Actual size of specimen = 3cm = 30,000 µm = 30,000 µm 260.13 µm = 115.33 x T.M.M. Cantillano Microscopy and Slide Preparations  Micrometry NOTE THAT: You must Calibrate the objectives first (Scanning, LPO, HPO, OIO) before preparing and measuring your specimens. Measure specimen using the most appropriate objective T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Wet Mount, Hanging drop, and Staining What is the principle behind Wet Mount and Hanging Drop Method? 1. Observe cell activities such as motility and binary fission. 2. Observe the natural sizes and shapes of the cells. T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Wet Mount T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Hanging drop T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining WHAT IS GRAM STAINING? Gram Staining is a differential staining procedure employed to differentiate Gram Positive bacteria from Gram Negative bacteria based on their cell wall. T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining VS. Gram Positive Gram Negative ✓ thick cell wall ✓ Thin cell wall ✓ Peptidoglycan is more ✓ Second lipid membrane called than 50% of dry weight lipopolysaccharides ✓ More of a gel than a rigid layer T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining Reagents used: 1. Crystal violet 2. Gram’s Iodine 3. 95% Ethanol 4. Safranin T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining T.M.M. Cantillano Microscopy and Slide Preparations  Slide Preparations: Gram Staining Bacillus subtilis Escherichia coli T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology WHAT IS MORPHOLOGY? In microbiology, the term morphology means cell shape. T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Bacteria T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Bacteria T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Bacteria T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Fungi T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Fungi T.M.M. Cantillano Microscopy and Slide Preparations  Microbial Cell Morphology: Fungi T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy I. Letter “e” mounting II. Wet Mount III. Hanging Drop Technique IV. Micrometry V. Gram Staining T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Materials Cut out Letter “e” Labeling materials Cut out thin leaf/ decaying fruit Cleaning materials Cake of baker’s yeast (for the Waste bottle whole class) Test tube and test tube rack Lagoon water (freshly collected) Alcohol lamp Distilled water (1L/ group) Wash Bottle NaCl Solution Ocular and Stage Micrometer (2 Yogurt pcs for the whole class) Toothpick Microscope Plastic dropper Stains (Crytal Violet, Gram’s Vaseline (Petroleum Jelly) Iodine, Safranin, Methylene Blue) Glass slides and cover slip T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure I. Each member should prepare a cut-out letter “e”, and place it at the center of a glass slide. With a plastic dropper carefully place a drop of water on the surface of a clean slide Cover the drop with a clean coverslip. View under 4x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure II. Prepare of thin leaf or a part of a decaying fruit and carefully place it at the center of the slides (prepare 2 slides for each group) Carefully drop a right amount of water and cover the it with a clean coverslip. View under 4x, 10x, and 40x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure III Prepare your lagoon water samples Prepare a clean depression slide/ improvised depression slides Using a toothpick, spread a thin ring of Vaseline approximately ¼ inch outside the concavity Transfer 2 loopfull at the central surface of the cover slip Invert the slide and center the depression over the droplet on the coverslip. Press lightly. Quickly turn over the slide View under 4x and10x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure IV Prepare your solution by adding water in a test tube with yeast enough to cause visible clouding (approximately 1 loopful per 15 ml of water) Remove a small amount of the suspension with a plastic dropper and carefully place a drop on the surface of a clean slide Cover the drop with a clean coverslip. View under 40x magnification Using ocular and stage micrometer, measure at least 5 cells (Calibrate the objectives first) T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure IV Put a droplet of methylene blue at the center of the slide Gently scrape the inside of your cheek with a clean cotton swab or toothpick. Transfer the sample onto the center of a clean glass microscope slide by smearing the swab in a thin layer. Carefully put a clean coverslip. View under 40x magnification Using ocular and stage micrometer, measure at least 5 cells (Calibrate the objectives first) T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Wet Mount, Hanging Drop and Gram Staining Procedure Prepare your samples. Add a smear on a clean slide Heat fix the bacterial samples Add stains View under 4x, 10x, and 40x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Laboratory Activity 1: Microscopy Expected Output Picture of each slides that you prepared Microscopic image of cut out letter “e” under 4x magnification Microscopic image of your leaf/ portion of decaying fruit under 4x, 10x, 40x magnification Microscopic image of “suspected algae” in your lagoon water samples under 4x and 10x magnification Microscopic image of yeast/ cheek cell under 40x magnification. Calibration constant of Scanning, LPO and HPO Measurement of 5 yeast/ cheek cells under HPO Microscopic image of gram-stained cells under 4x, 10x, and 40x magnification T.M.M. Cantillano Microscopy and Slide Preparations  Polytechnic University of the Philippines College of Science Department of Biology Microbiology and Parasitology (Laboratory) Laboratory Discussion VI: Macroscopic and Microscopic Examination of Stool Sample Trisha Mae M. Cantillano  Overview Why do you need to gain skill in stool examination? T.M.M. Cantillano  Stool Analysis A stool analysis is a series of tests done on a stool sample to help diagnose certain parasitic infections like Ascariasis, Trichuriasis, and Schistosomiasis T.M.M. Cantillano  Stool Sample Stool sample can be used to diagnose Collection or rule out a health condition. Stool may contain parasites such as Ascaris lumbricoides and Giardia lamblia that cause intestinal infection Why is it done?? Ascaris lumbricoides Giardia lamblia T.M.M. Cantillano  Stool Sample 1. Label a clean, screw-top container Collection with your name, date of birth, and the date of collection. Please see sample of the container below. How is it done? Screw cap container T.M.M. Cantillano  Stool Sample 2. Place something in the toilet to catch Collection the stool sample, such as an empty plastic food container, or spread clean newspaper or plastic wrap over the rim of the toilet. How is it done? 3. Make sure the stool does not touch the inside of the toilet. 4. Use the spoon or spatula that comes with the container OR clean popsicle stick to collect the stool sample. T.M.M. Cantillano  Stool Sample 5. Put the fecal sample inside the Collection container and then screw the lid shut. 6. Aim to collect a stool sample about the size of a walnut. How is it done? 7. Put anything you used to collect the stool in a plastic bag, tie it up and put it in the bin. 8.Wash your hands thoroughly with soap and warm running water. T.M.M. Cantillano  Stool Sample The stool sample must be fresh when Storage examined with parasites to avoid false diagnosis. A fresh stool sample is still soft and moist within the last 4-6 hours. How is it stored? Put the container with stool sample inside a Ziplock. If stool sample cannot be examined right away put it in a fridge in the Microbial and Parasitological Laboratory. Do not put it in a fridge in your kitchen. T.M.M. Cantillano  MACROSCOPIC Examination of Stool Samples Note the color of the specimen (Dark brown, brown, pale brown, red-brown, yellow, clay- colored, green, black) Note the consistency of the stool. Mushy or liquid stools suggest the possible presence of trophozoites or intestinal protozoa. Protozoan cysts are found most frequently in formed stools. Helminth eggs and larvae may be found in either liquid or formed stools. Use Bristol Stool Chart to describe the consistency of the stool. Examine the stool for blood or mucous – Fresh blood appears bright red – this indicates acute lower intestinal tract bleeding Bloody mucus suggests ulceration Break up the stool with applicator stick to check for the presence of adult helminth like Ascaris. T.M.M. Cantillano  Microscopic Examination of Stool Samples Wet mount procedure T.M.M. Cantillano  Microscopic Examination of Stool Samples Stained Slide Preparations 1. Prepare a thin even smear of the material by streaking the material back and forth on the slide with an applicator stick. (If necessary, dilute feces with saline) 2. Apply one drop of Lugol’s iodine and mix thoroughly with stick applicator. 3. Place the cover slip 4. Systematically examine the smear microscopically. T.M.M. Cantillano Normal stool findings Stool plant cells, plant, and hairs Pollen grains Undigested Citrus fruits Stool starch and iodine stain of starch result Gram negative bacteria Stool showing potato remnants squamous epithelial cell and macrophagic cell Polymorphonuclear leucocytes  Stool Sample Disposal Dig a pit 4-5 meters deep and 1-2 meters wide Make a lid that fits tightly over the pit. Throw stool into the pit and replace the lid immediately. Once a week cover the refuse with a layer of dried leaves T.M.M. Cantillano  Laboratory Exercise 5 tt Stool Sample Examination Lab Apparatus to To Prepare Materials to bring – Fresh Stool Sample (3 Request (each group) samples) – Screw cap container – (1) Microscope – Normal Saline Solution – 15 ml conical tubes – (1) Alcohol lamp – Cleaning materials – Disposable droppers – (1) Test-tube Rack – Applicator stick – (1) Wash bottle – Lighter/ Matchstick – Glass slides Chemicals to Request – Cover slips (whole class) – Blue sharpie – Lugol’s Iodine – Scratch paper (with – Crystal Violet printed letters)/ newspaper – Gram’s Iodine – Waste bottle – Safranin T.M.M. Cantillano  tt End- T.M.M. Cantillano  Polytechnic University of the Philippines College of Science Department of Biology MICROBIOLOGY AND PARASITOLOGY (LABORATORY) BIOL 014 Laboratory Discussion 5: Observation of Yeast and Molds Trisha Mae M. Cantillano  Yeast and Molds YEAST are UNICELLULAR fungi MOLDS are MULTICELLULAR with filamentous structures T.M.M. Cantillano Observation of Yeast and Molds  Significance of Yeast and Molds ❑ Role in decomposition and nutrient cycling ❑ Industrial applications ❑ Yeasts: Fermentation (e.g., baking, brewing) ❑ Molds: Production of antibiotics, enzymes, and organic acids ❑ Pathogenic impacts on humans and plants T.M.M. Cantillano Observation of Yeast and Molds T.M.M. Cantillano Observation of Yeast and Molds  Microbial Cell Morphology: Fungi T.M.M. Cantillano Microscopy and Slide Preparations T.M.M. Cantillano Observation of Yeast and Molds  Figure 1. Curvularia sp. PUPML_2020227 isolate from watermelon (A) Observed (B) reverse (C)-(E) Conidiophores with an integrated conidiogenous cell producing immature conidia (F)- (M) straight to ovoid septate. How to Describe? The colony of the 7-day old culture of the isolate PUPML_2020227 grown on potato dextrose agar (PDA) at 25 ⁰C appeared to be off-white with light olive green color and pale brown at the center; mycelial growths are powdery-cottony with filamentous margin, and slightly elevated (Figure 1A- B). Conidiophores were unbranched, sub-hyaline to pale brown, flexuous to straight and septate (Figure 1C-F). Conidiogenous cells were integrated, terminal and intercalary. The observed conidia were ellipsoidal to obovoid, asymmetrical with hilum, appeared pale brown to brown in color, usually curved at the third cell from the base, and 2-3 distoseptate (Figure 1G-M). T.M.M. Cantillano Observation of Yeast and Molds T.M.M. Cantillano Observation of Yeast and Molds T.M.M. Cantillano Observation of Yeast and Molds  Laboratory Activity 4: Isolation of Fungal Specimens from Spoiled Fruit/ Vegetables Expected Output MACROGRAPHS (Pictures of the plates from 0-7 days, Observed and Reverse) MICROGRAPHS (Pictures of your suspected fungal agent before inoculation on the media; under 4, 10, and 40x magnification) Table of the measured diameter (mm) per day (of each replicates) Graph of the growth rate of your fungal specimen Laboratory Report and Lab Entry on Lab notebook T.M.M. Cantillano Microscopy and Slide Preparations -Sub-Activity  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 (