Lab Safety BSCI223 Lab Midterm Study Guide Pathogens microbes that are capable of causing disease Biosafety level explains the pathogenicity of microbes studied in the lab and dictates the precautions that must be taken to ensure the safety of the lab worker BSL 1 the microbe is not known to cause infectious disease BSL 2 the microbe is slightly associated with human disease BSL 3 Indigenous or exotic agents with potential for aerosol transmission disease may have serious or lethal consequences BSL 4 dangerous exotic agents which pose a high risk of life threatening disease aerosol transmitted lab infections or related agents of unknown risk or transmission The likelihood of a microbe causing disease depends on the characteristics of the microbe the characteristics of the host and exposure to the infectious agents Microbes will only cause disease if they enter into the system at an appropriate site a portal of entry Reservoir all of the places where an infectious agent may be found Goal of standard practice to make sure that microbes are not transmitted from their reservoirs to a human worker Universal Precautions a set of guidelines for the handling of all blood and body fluids All body fluids are treated as potential sources for infectious agents Microbes can enter the body via cuts so gloves must be warn There is no eating drinking or smoking in a lab where body fluids are handled Hands are always washed No open toed shows and lab coat must cover lap Goggles must be warn when there is a risk of aerosolizing culture flaming cultures and use of vortex mixer Long hair must be tied back Aseptic technique Lab benches must be scrubbed with disinfectant at the beginning and end of each lab Microscope eyepieces must be wiped with alcohol Goggles must be disinfected after each lab Do not chew gum pens or fingers Do not rub eyes Wash hands with soap and water after each lab Leeuwenhoek s Beasties simple microscope Leeuwenhoek discovered microbes under his own handmade Wavelength the human eye sees visible light through the electromagnetic spectrum Magnification the apparent increase in size of an object This is limited by resolution Resolution the ability to distinguish between objects that are close together It is a function of the wavelength of the light source and the numerical aperture of the lens Visible light has a wavelength range of 400 700 The smallest objects that can be resolved by the light microscope are in the range of 0 2 micrometers Numerical aperture the light capturing ability of the lens It is a function of the sin of the angle theta To fix the issue of refractive index oil is used on high magnification Limit of resolution distance of two points which can be seen as distinct Contrast the differences in intensity between two objects or the object and its background It is produced in the specimen by the absorption of light brightness reflectance birefringence light scattering diffraction fluorescence or colorations Increasing contrast decreases resolution Bright field microscope light microscope used for visualization of stained specimens includes a compound microscope which is what we use in the lab Light passes through the condenser lens to the specimen and on to the objective and ocular lenses Arm supports the body and is the part to grasp when carrying Body tube connects the ocular lens with the nosepiece Nosepiece rotating part to which the three objective lenses are the microscope attached Objectives lenses that magnify images 4x 10x 40x 100x The 100x objective uses oil immersion to view a specimen Ocular lens lens that magnifies the real image another ten times The actual magnification is 10x what the objective lens says because of the ocular lens Stage supports the microscope slide Condenser concentrates the light before it passes through the specimen Iris diaphragm regulates the amount of light entering the condenser Coarse adjustment brings the specimen into initial focus Moves the stage up and down Fine adjustment brings the specimen into final focus Dark field microscope light microscope used for viewing live unstained specimens that are motile or have an unusual shape Light passes through a condenser modified with a dark field stop which blocks incident light from going to the condenser and results in a dark field Light only passes onto the specimen Phase contrast microscope light microscope that uses light for viewing live specimen that would be damaged or altered by attaching them to slide or staining them Light passes though a diaphragm that modifies the light into a hollow cone It increases the contrast between the specimen and background Electron Microscope can resolve images in the nanometer range and view molecules and cell structures that are less than 0 2 micrometers Three domains eukarya bacteria and archaea Eukaryote Microbes Protozoa unicellular non photosynthesizing lack a rigid cell wall Ex amoeba and paramecia Fungi unicellular or multicellular lack chlorophyll and usually bear spores Can be filamentous such as aspergillus niger They also may use budding for reproduction Ex yeast mushrooms Algae unicellular or multicellular perform photosynthesis and lack tissue differentiation Ex chlorella Prokaryote Microbes Bacteria cell walls with peptidoglycan Archaea cell walls with pseudopeptidoglycan usually live in very extreme environments A rigid cell structure indicates that the cell has a cell wall Aseptic Technique Streak plates to produce well separated colonies of bacteria Use an inoculating loop to dilute the bacteria into colonies Colony a mound of bacteria on a streak plate A streak plate of mixed culture will show colony types of each culture To isolate a pure culture you pick one phenotype of colony to restreak Refrigeration is used for short term storage of culture 80 degree freezers and lyophilization are used for long term storage of culture Things to look for in a well isolated colony Colony diameter Pigmentation Form elevations and margin Smooth rough or mucoid surface Opacity translucent transparent or opaque Texture Staining colored Stains used to increase contrast Salts in which one of the ions is In a basic stain the color is usually positively charged In an acidic stain the color is usually negatively charged Direct or Positive Staining Procedure cell takes up a positively charged die and becomes stained Ex CV methyl blue safranin Indirect or Negative Staining Procedure cell is immersed in a negatively charged die The cell