Lab 1 - Preparation Slides PDF

BIOL 2P98 Lab 1 Introduction and Microscopy Binomial nomenclature • Bacteria as with other organisms are named with two identifying names: first is the Genus name and the second is the species name. • Genus is always capitalized and species begins with a small letter. • The Genus and species are e...

BIOL 2P98 Lab 1 Introduction and Microscopy Binomial nomenclature • Bacteria as with other organisms are named with two identifying names: first is the Genus name and the second is the species name. • Genus is always capitalized and species begins with a small letter. • The Genus and species are either individually underlined or italicized. • For example: Escherichia coli or Escherichia coli • Notice the break in the underline between Genus and species. Aseptic Technique procedure • Wearing your PPE (Lab coat, tight fitting gloves and safety glasses) wipe down your benchtop with 70% ethanol using squirt bottle and paper towel. • Label your plate with your name, AT (for Aseptic Technique), Lab section, date and media type (TSA for Tryptic Soy Agar). • Light your Bunsen burner with the flint striker and adjust the amount of air and gas to achieve an inner and outer cone of flame. • Hold the inoculating loop handle like you would a pen with the loop down. • Sterilize the loop starting with the wire closest to the handle, working your way down towards the loop end, making sure each section is bright red before moving. This method will reduce or eliminate the formation of aerosols by allowing the loop to heat up slowly. • Do not shake your loop or set it down while it cools. Just hold it steady out of the flame for about 10 seconds. • With your other hand pick up a test tube of sterile water from the test tube rack. Use the pinky of the hand with the loop to slowly remove the lid off the test tube. Keep the pinky curled around the lid, do not place down on the bench top. • Pass the neck of the open tube quickly through the flame 3 times to sterilize the air in and around the mouth of the tube. • Insert your sterile loop into the tube to collect a sample. • Withdraw the loop slowly not touching the sides or mouth of the tube. • Pass the neck of the tube through the flame quickly 3 times and then replace the lid back on the tube and set back into the test tube rack. • Still holding the loop, use the opposite hand to lift the small agar side of the plate. Inoculate with a continuous streak onto the agar of the plate. Agar is soft and can tear with too much pressure, use a gentle pressure to glide over the agar surface. • Re-flame the loop to sterilize and then set onto foil provided at the bench. • Seal the plate with Parafilm to keep the lid on and moisture in. It will be incubated upside down to prevent the condensations from touching the agar surface. • With proper aseptic technique there should be no growth on the plate inoculated with sterile water. Aseptic Technique • Create a zone of sterility Use top of inner cone to sterilize Aseptic transfer of a liquid culture Madigan et. al., 2021, p. 113 Labeling and Incubation of Plates • Plastic petri dishes are labeled on the bottom (agar side) because the lid can • • • • • be misplaced and label around the outer edge to keep the centre area open for observations. Glass tubes or glass culture dishes should be labeled using a piece of scientific coloured tape and write the information on the tape. Need to label the name of the organism, the type of media and the date as minimum information. Also for our labs, include your group number/name, lab section, test parameters. Label before you inoculate. Parafilm is used to hold the lid onto the bottom agar section, allows for some air exchange, but prevents contaminants from getting in and your culture from getting out. Plates are incubated and stored upside down to prevent the condensation from touching the agar surface. Isolation of a Pure Culture Introduction • Microbial populations usually exist as mixed populations. • To study individual species, bacteria must be isolated into a pure culture. • A pure culture is a population of identical cells derived from a single cell. • There are four main methods for isolating a pure culture: Quadrant Streak plating, Pour Plate, Spread Plate and Filtration. Madigan et. al., 2021, p. 2 Isolation of a Pure Culture • Quadrant Streak Method • Pour Plate Method • Spread Plate Method Talora & Talora, 2002, p. 63 Quadrant Streak Method • • • • Flame loop Collect sample Streak Quadrant 1 Flame loop Flame loop Touching Touching Touching Quadrant 3 Quadrant 1 Quadrant 2 spread to make spread to make spread to make Quadrant 4 & Quadrant 2 Quadrant 3 thru middle Leboffe, 2002, pg 21 Madigan et. al., 2021, p.113 Pour Plate Method • For this method you will want to perform a serial dilution with your loop either within the molten agar (cooled enough not to kill organisms); or in a sterile broth tube. Vortex in between taking a sample with your loop to the next tube dilution. • Once you have your dilution tubes ready, you will want to pour the molten agar into the empty sterile petri dish; or add a small amount of broth culture into the dish and pour the molten sterile agar into the petri dish. Swirl gently to distribute the molten agar in the dish without splashing onto the lid. • Allow the molten agar to solidify before Parafilming the plates and then incubating. Madigan et. al., 2021, p.115 Spread Plate Method • For this method you will want to perform a serial dilution in sterile broth tubes. Vortex in between taking a sample with from one tube to the next tube dilution. This can be done by pipetting from one tube to the next. • You will then pipet a small volume onto already prepared agar plates. • Using a sterile spreader then evenly spread the small broth volume around the surface of the agar plate. • Parafilm the plates and incubate. Madigan et. al., 2021, p.115 Physical states of Media • Solid agar can be poured into tubes or plates before solidifying. The media contains 1% to 1.5% agar. • Semisolid agar tubes contain a lower percentage of agar. This media allows for motile organisms to move about, also while reducing the movement of gasses such as oxygen, providing an anaerobic environment. • Broth tubes are liquid and do not contain any agar. Colony Morphology or Cultural Characteristics on agar plates • Pigmentation (Colour) • Non-pigmented • Cream, Tan, Beige, White • Pigmented • Red, Yellow, Green, Black • Orange, Purple, Pink • Optical Characteristics • Opaque, Transparent, Translucent Leboffe & Pierce, 2011, p. 20 Growth patterns on Agar slants Leboffe & Pierce, 2011, p. 27 Brown, 1998 Growth in broth tubes Leboffe & Pierce, 2011, p. 27 Microscopy Zeiss Primo Star • • • • • • • • • • • • • • • • • 1 Eyepieces 4 Carrying handle 5 Plug-in Power unit 6 Illumination-Intensity indicators 7 Rotary knob for On/Off & Adjusting Intensity 8 Fine Focusing Drive 9 Coarse Focusing drive 10 Control knob for X travel 11 Control knob for Y travel 12 Clamping screw for condenser DO NOT TOUCH!! 13 Transmitted-light illuminator 14 Luminous field diaphragm 15 Centering screws for condenser DO NOT TOUCH!! 16 Abbe condenser DO NOT TOUCH!! 17 Objective 18 Microscope Stage 19 Spring level of specimen holder Objective lenses • Magnification (i.e. 10X) • Numerical aperture (i.e. 0.25) • Multiply by 1000 for maximum magnification to keep proper resolution • OIL for Oil Immersion Calibrating the Ocular Micrometer (ruler in the eyepiece) • Line up the zero’s of the stage micrometer and the ocular micrometer • Find another place farther down the rulers where they both line up again 15 ocular 10 stage 10 stage divisions x 10 micrometer per stage division = 6.67 micrometers per ocular division 15 ocular divisions Negative Stain • For bacteria too sensitive to be heatfixed or for determining accurate size • Acidic dye with a negative charge, is repelled by bacteria, stains the background • Nigrosin, an acidic dye, is used in our lab Leboffe, 2002, p. 85 Cellular Morphology Cell Shape and Arrangement Shape: Bacillus (rod shape) Shape: Coccus (spherical shape) Alexander & Strete, 2000, pg 14 Shape: Spirillum (Spiral shape) Leboffe & Pierce, 2011, p. 42 Kaiser, 2005 Calculating the Size of a Cell • Once you know the length of each ocular division of the microscope you are using, then you can easily measure and calculate the size of a single cell in your view. • You count how many ocular divisions in length your cell is and then multiply by the length of one ocular division. • Length in # ocular divisions x mm per ocular division = cell length in mm Example: spirillum shape cell in image has a length of 16 divisions. Using the 100X objective, we had already calculated the length of one division to be 1 mm. Therefore the length of the spirillum cell is 16 x 1 mm = 16 mm References • Alexander SK and Strete D (2000) Microbiology A Photographic Atlas for the Laboratory. Redwood City, Calif.: Benjamin/Cummings. • Brown AE (1998) Benson’s Microbiological Applications Laboratory Manual in General Microbiology. (10th Ed). Dubuque, IA: J. Roerig-Blong Publishers. • Kaiser GE (2005) Bio 230 Microbiology Lab Manual. Retrieved June 17, 2005 from http://www.cat.cc.md.us/courses/bio141/labmanua/toc.html • Leboffe MJ (2002) Microbiology Laboratory Theory and Application, 2nd edition. Morton Publishing Company, Colorado. • Leboffe MJ and Pierce BE (2011) A Photographic Atlas for the Microbiology Laboratory, 4th edition. Morton Publishing Company, Colorado. • Madigan MT, Bender KS, Buckley DH, Sattley WM, and Stahl DA (2021) Brock Biology of Microorganisms, 16th edition. Pearson Education, Inc., New York. • Talora KP and Talora A (2002) Foundations in Microbiology Basic Principles. (4 th ed). New York, New York: J. Smith Publisher.